Method for detecting and analyzing protein interactions in vivo

ABSTRACT

Described are various methods of detecting and analyzing protein interactions in a cell. The specific protein interaction is detected by a permanent signal generated by a recombinase activity or protease activity that depends on the protein interaction.

This application is a national phase entry of PCT Application No.PCT/EP03/02611, filed on Mar. 13, 2003, which claims the benefit under35 U.S.C § 119(a)-(d) or (f) of German Patent Application No. DE1021063.8, filed on Mar. 13, 2002, each of which is incorporated byreference herein in its entirety including all figures and tables.

FIELD OF THE INVENTION

The invention relates to various methods of detecting and analyzingprotein interactions in a cell, which methods involve the appearance ofa specific protein interaction being converted to a permanent detectionsignal by means of providing, in a manner dependent on said proteininteraction, a recombinase activity or protease activity. The detectionsignal is amplified here, compared to reporter gene activation by way ofclassical transcriptional activation of reporter genes.

The invention furthermore relates to cells selected from the groupconsisting of bacteria and yeast cells and of cells of higher eukaryoticcell lines from the group consisting of rodents and Homo sapiens, whichcells have been transfected, either stably or transiently, with at leastone expression vector, comprising at least one construct for expressingthe components indispensible for carrying out the method of theinvention.

The invention furthermore relates to a kit for carrying out theabovementioned methods.

The method of the invention in particular makes possible detection ofthe dynamics of specific protein interactions. Both the coming intobeing and the induced dissociation of interaction partners aredetectable. Said interaction partners may be in direct contact or may bepart of a protein complex. The method is particularly suitable fordetecting transient interactions, very weak interactions or thoseinteractions induced by a cellular stimulus or a substance.

BACKGROUND OF THE INVENTION

Virtually all biological processes in living organisms are controlled bythe function of specifically interacting proteins. Specific analysis ofprotein interactions makes it possible to isolate unknown proteins andto assign them to functional groups and also to elucidate the molecularmechanism of action of known proteins. Cellular signal transductionwhich comprises the transfer of extracellular signals to specificintracellular alterations is the key mechanism for controlling a cellduring development and in the response to environmental changes. Thetransfer of these signals is controlled by strictly regulated cascadesof specifically interacting proteins. In addition, virtually allimportant cellular functions which are usually coupled to signaltransduction are carried out by controlled protein interactions (Pawsonand Scott 1997). These include, inter alia, control of the cell cycle,protein synthesis and protein degradation, prevention or induction ofapoptosis, transport processes, detection and induction of stimuli, geneexpression, mRNA processing, DNA synthesis and DNA repair and the entireenergy metabolism. All these processes are very dynamic, i.e. they aresubject to alterations which are embedded in the overall state of thecell. This is reflected, at the protein level, by the regulatedalteration of the composition of the function-performing complexes(Ashman, Moran et al. 2001).

Proteins and their interactions are subject to great and dynamicalterations in the cell which are frequently brought about by signaltransduction cascades. Their function or the composition of proteincomplexes may also change due to allosteric effects or a change ofintracellular location. The regulation of the function of proteins isparticularly dependent on activation by enzymes of the signaltransduction cascades which catalyze protein modifications on specifictarget proteins. Post-translational modifications often have a dramaticinfluence on the activity of a multiplicity of proteins. Regulatedprotein phosphorylation, protein acetylation, protein methylation,protein sulfation, protein acylation, protein prenylation, proteinribosylation, protein glycosylation, protein ubiquitination orproteolytic activation and inactivation are known modifications some ofwhose effects and regulatory mechanisms are only little understood.Modification-dependent protein interactions have been described for amultiplicity of transduction pathways in different cellular adaptationevents (Hunter 2000).

The activation or deactivation of particular signal transductioncascades must be exactly controlled in the cell, both by way of time andits form. A multiplicity of pathological processes in cells is caused byinterference in the control of signal transduction and may lead tometabolic disorders, cancerogenesis, immunological disorders orneurological deficits. Pathological processes of this kind may be causedin particular by specific mutations in the genes coding for proteinshaving an important function in neuronal signal transduction. Thus, forexample, particular mutations in the genes of NMDA receptor subunitsalter the composition and signal properties of the NMDA receptor proteincomplex (Migaud, Charlesworth et al. 1998). Molecular mechanismsresponsible for the precise time control of cellular events and oftencontrolled by feedback mechanisms are only beginning to be understood(Marshall 1995). Time-limited protein-protein interactions, controlled,for example, by reversible post-translational modifications exert a keyfunction here (Yasukawa, Sasaki et al. 2000)(Hazzalin and Mahadevan2002).

Currently there is a lack of suitable methods for identifying andanalyzing regulated, time-limited or modification-dependent proteininteractions.

A multiplicity of methods of characterizing protein-protein interactionshave been described.

Biochemical methods of protein purification, coupled tomass-spectrometric analytical methods, enable protein complexes to becharacterized (Ashman, Moran et al. 2001). Thus it was possible, forexample, to isolate, with the aid of a tandem affinity purification(TAP) (Rigaut, Shevchenko et al. 1999), denaturing one-dimensional gelelectrophoresis and tryptic digestion of single bands in combinationwith mass-spectrometric analysis, to isolate a multiplicity of proteincomplexes from yeast and to determine the components thereof (Gavin,Bosche et al. 2002). Using an optimized immunoprecipitation protocol,mass spectrometry and the Western blot technique, a multiplicity of thecomponents of the neuronal NMDA receptor signal processing complex wascharacterized (Husi, Ward et al. 2000). These biochemical-biophysicalmethods seem particularly suitable for the analysis of stable and staticprotein complexes, but have a few experimental disadvantages anddisadvantages in principle. Thus, all biochemical methods areexperimentally very expensive and require a very large amount ofbiological starting material. Moreover, already optimized purificationmethods (e.g. the TAP method) require the corresponding fusion proteinsto be transgenically and stably expressed in the organisms of choice. Inthe analysis of complex tissues, weakly expressed or celltype-specifically expressed proteins may readily be below the limit ofdetection. A fundamental disadvantage of all biochemical methods arisesfrom the necessity of cell disruption or of the solubilization of largemembrane complexes. Weak or transient interactions may readily be lostduring the course of the biochemical workup which usually requires aplurality of steps (Ashman, Moran et al. 2001). Analyses of theinteractions of proteins with extreme physicochemical properties, suchas, for example, membrane proteins or proteins having a high totalcharge, in each case need an optimized protocol or, in individual cases,are not possible.

Previous mass-spectrometric methods have only limited suitability forcharacterization of post-translationally modified proteins, sincemodifications such as specific phosphate radicals may readily be lostduring fragmentation (Ashman, Moran et al. 2001). The composition andfunctional activity of protein complexes are subject to constantdynamics and are controlled by a multiplicity of regulatory mechanisms.Moreover, it became clear recently that specific physicochemicalproperties or specific environments, such as specific membranetopologies and membrane compositions for example, strongly influenceprotein-protein interactions. Thus, for example, the lipid compositionof the different intracellular membrane systems plays a large part inthe assembly of protein complexes (Huttner and Schmidt 2000). Owing totheir particular molecular composition, “lipid rafts”, subdomains in thecell membrane, allow completely different interactions than neighboringregions in the lipid bilayer (Simons and Ikonen 1997). Biochemicalmethods have only limited suitability for detailed analysis of thespecific interactions or interaction domains of proteins within acomplex or for analysis of possible transient interactions of associatedproteins, due to the relatively high experimental costs.

Microarrays are another tool for systematically analyzingprotein-protein interactions, protein-peptide interactions or theinteractions of proteins with low molecular weight substances. Thismethod involves applying in-vitro translated or recombinantly producedproteins or peptides, antibodies, specific ligands or low molecularweight substances to a support material, analogously to DNA microarrays.Complex protein mixtures or substance libraries may thus besimultaneously screened, inter alia, for specific interactions (Ashman,Moran et al. 2001)(Xu, Piston et al. 1999). However, the analyses usingprotein or substance arrays, which are carried out completely in vitro,likewise have great disadvantages. Thus, proteins are prepared oranalyzed under completely artificial conditions in this method.Furthermore, the appearance of high unspecific background signals, thelimited sensitivity and difficulties in detecting proteins havingparticular physicochemical properties greatly limit the number ofpossible applications of these array-based in-vitro analytic methods fordetecting and analyzing protein interactions (Ashman, Moran et al.2001).

There are furthermore various methods known which involve detectingprotein interactions in the cell, and thus in vivo, by indirectlyactivating genetic reporters. Most of the familiar methods are limitedto detection of binary protein interactions in karyoplasma and are basedon the functional modularity of transcription factors, such as the2-hybrid system in yeasts, for example (Fields and Song 1989). The2-hybrid system involves expressing in yeast cells one or more proteinsor protein sections as fusion proteins with a DNA binding proteinwithout transactivation capability and using them as “bait” fordetecting interacting components. A second protein or protein fragmentis expressed as fusion protein with a transcriptional transactivationdomain and is the “prey” component. The “prey” component frequently is afusion protein which comprises a gene product of a complex cDNA libraryin addition to the transcriptional transactivation domain. Theinteraction of the bait and prey fusion proteins results in functionalreconstitution of the activating transcription factor. The reportergenes used in the 2-hybrid system are enzymes which can be used todetect said protein interaction either by growth selection or by asimple colorimetric assay and which are very sensitive. A reporter genefrequently used in the 2-hybrid system; which makes possible a positivegrowth selection of cells with specific protein-protein interactions, isthe histidine 3 gene which is an essential enzyme for histidinebiosynthesis and whose protein-protein interaction-dependent expressionenables the cells to grow on histidine-deficient medium. The mostfrequently used reporter gene whose protein-proteininteraction-dependent expression is detectable by a simple colorimetricassay is the beta-galactosidase gene.

The 2-hybrid system was originally developed in yeast, but subsequentlyvariants of the 2-hybrid system have also been described for applicationin E. coli and in higher eukaryotic cells (Luban and Goff 1995)(Karimova, Pidoux et al. 1998) (Fearon, Finkel et al. 1992) (Shioda,Andriole et al. 2000).

A substantial disadvantage of classical 2-hybrid-based systems is, interalia, the relatively high rate of false-negatively and false-positivelydetected interaction partners. This is due, on the one hand, to the highsensitivity of the reporters, but also to spatial coupling of theinteraction and the basal transcription machinery. Recently, interactionsystems for yeast cells have been described, which spatially decouplethe place of interaction from the activation of the reporter genes usedor the selection mechanisms used for detection (Maroun and Aronheim1999). Related systems in yeast also allow at least one interactionpartner which may be an integral or membrane-associated protein to beanalyzed (Hubsman, Yudkovsky et al. 2001) (Ehrhard, Jacoby et al. 2000).

The functional complementation of proteins or enzymes which is also thebasis of classical 2-hybrid-based systems is a method of analyzinginteractions in living cells and bacteria, which has been known andapplied for some time (Ullmann, Perrin et al. 1965) (Fields and Song1989) (Mohler and Blau 1996) (Rossi, Charlton et al. 1997) (Pelletier,Campbell-Valois et al. 1998). Transcomplementation means the separationof an intact and functional protein or protein complex into twoartificial subunits at the gene level. The two subunits here are per seinactive with respect to the function of the complete protein but areactive with respect to their corresponding subunit function and areincapable of self-reconstitution. By providing in a proteininteraction-dependent manner close spatial proximity between the twoseparated subunits, the fusion of such subunits to proteins or proteindomains interacting with one another results in complementation of thedivided protein, thereby rendering it functional again. The regaining ofthe function of the protein (e.g. an enzyme) by protein interaction isutilized here directly or indirectly for detection of said interaction(Mohler and Blau 1996) (Rossi, Charlton et al. 1997). The best-knownexample is transcomplementation of the transcription factor Gal4 whichis the basis of the classical yeast 2-hybrid system (Fields and Song1989).

In addition, transcomplementations and, coupled thereto, methods ofdetecting protein interactions have been described for differentproteins with enzymatic activity, inter alia beta-galactosidase (bGal),dihydrofolate reductase (DHFR) and beta-lactamase (bLac) (Michnick andRemy 2001) (Rossi, Charlton et al. 1997) (Michnick and Galarneau 2001).Protein interactions can be detected indirectly in these systems aftertranscomplementation of the abovementioned enzymes by way of growthselection or of fluorimetric or colorimetric enzyme detection assays.Depending on the substrate used, detection may usually be carried outonly after disruption of the cells and addition of the substrate invitro.

In contrast, the DHFR-based system enables the interaction ortranscomplementation of proteins to be detected also in vivo. For thispurpose, a cell-permeable fluorescently labeled antagonist(methotrexate) is added which binds only to the intact protein. Thedisadvantage here, however, is the fact that the antagonist is not asubstrate of the enzyme but binds the enzyme as a competitive inhibitor.Therefore no enzymatic enhancement of the detection signal whatsoever isproduced. This results in a strong reduction in the sensitivity of thisdetection method compared to detection of positive clones by way ofpositive growth selection under the appropriate culture conditions.Moreover, the detection signal in the DHFR-based system for analyzingprotein interactions is visible only directly after addition of thefluorescently labeled inhibitor, i.e. it is not possible to detectdynamic processes in cells, which are frequently accompanied by dynamicor transient protein interactions, by means of these nonpermanentsignals which are detectable only for a short time. In addition, theinhibitor described (methotrexate) is a highly cytotoxic substancewhich, after application, greatly impairs and alters cell growth,metabolism and other intracellular processes and thus distorts thenormal in vivo conditions.

Although a DHFR-based method of analyzing protein interactions, which isbased on positive growth selection of cells by transcomplemented DHFR,and not on application of methotrexate, offers correspondingly highersensitivity, it requires periods of several days and weeks, a fact whichmakes this method appear not particularly suitable for application inhigh throughput methods, for example in high throughput screening. Owingto these disadvantages, these systems are also of only very limitedsuitability for analyzing transient or stimulus-induced interactions,since short-time transcomplementation is insufficient in order to enablepositive cells to be selected by growth over a longer period. On theother hand, binding of fluorescent antagonists such as methotrexate bythe transcomplemented DHFR can be detected only when finding the exactmoment of the transient or stimulus-induced interaction.

These problems in detecting transient, i.e. time-limited, proteininteractions also relate to the classical 2-hybrid system and to itsvariants known according to the prior art, since here too reportersystems are used which do not produce any permanent detection signal.

Only when they appear do transient protein interactions result inshort-time expression of the reporter genes used, with the gene productsof previously used reporter genes being able to generate a detectionsignal only during their lifetime, meaning that, for the 2-hybrid systemand its variants, and in particular for the analysis of weak andtransient protein interactions, only a relatively short time isavailable for detecting the protein interaction-dependent signals. Thisis a big problem, in particular when a large number of differentpotential interaction partners of a bait protein need to be testedsimultaneously for interaction, as in screening methods, in particularin high throughput screening methods, for example. Screening methodsmust enable a multiplicity of different potential interactions whichpossibly may also take place sequentially and possibly possess differentstrengths of interaction and lifetimes to be analyzed simultaneously ata defined point in time. However, if particular detection signals aredetectable only in a narrow “time window”, which possibly do not evenoverlap for different interactions to be detected, the 2-hybrid system,its variants and the known transcomplementation-based detection systemsfor analyzing protein interactions may not be able to record certaininteractions, in particular weak and transient protein interactions.

Another selection system which is based on a specific type of proteintranscomplementation is the split ubiquitin system originally developedfor studies in yeast and applied recently also in mammalian cells. Thissystem utilizes the separation of ubiquitin into two nonfunctionalmoieties, an N- and a C-terminal fragment (Nub and Cub) (Johnsson andVarshavsky 1994) (Rojo-Niersbach, Morley et al. 2000). Ubiquitin is asmall protein which labels proteins typically fused to its C terminusfor cellular degradation. This biological mechanism is used in the splitubiquitin system for detecting protein interactions. In one embodimentof the split ubiquitin system, a first fusion protein comprising theC-terminal fragment Cub, a selection marker protein or fluorescentprotein coupled thereto and a first interaction partner, and a secondfusion protein comprising the N-terminal fragment Nub and the secondinteraction partner are heterologously expressed in the cell. A specificinteraction of the corresponding fusion proteins restores a correctlyfolded ubiquitin which the proteasome can detect and process, with thecoupled, initially active reporter then being degraded. Accordingly, thesystem allows negative growth selection focussed on the absence of theselection marker or observation of the disappearance of a fluorescentreporter. These embodiments of the split ubiquitin system, described inscientific publications, thus disclose two very weak points, firstlynegative selection making rapid and unambiguous detection of relevantinteractions difficult and secondly no signal increase taking place inthe cell after the interaction. The latter point makes it virtuallyimpossible to detect weak or transient interactions.

The patent WO 95/29195 discloses an embodiment of the split ubiquitinsystem in which two different fusion proteins which comprise in eachcase one interaction partner and one part of the ubiquitin are expressedin a cell. One of the two fusion proteins here furthermore comprises areporter protein which can be proteolytically removed by aubiquitin-specific protease. Said reporter protein is removed here by aubiquitin-specific protease only after a specific protein-proteininteraction has occurred, and only then is activated. However, thisembodiment of the split ubiquitin system does not overcome the fact thata reporter molecule can be released or activated only once per eachinteraction which has taken place. The latter makes it virtuallyimpossible to detect weak or transient interactions. In addition, thereporter is coupled directly to one of the interaction partners, meaningfirstly that the amount of reporter strongly depends on the level ofexpression and the stability of the interaction partner to which it hasbeen fused. This leads to the possibility of an unstable or readilydegradable protein delivering a false-positive signal in the analysis.

Another system, described for mammalian cells, is based on activationand dimerization of modified type I cytokine receptors (Eyckerman,Verhee et al. 2001). Activation of the STAT3 signal pathway in thissystem can only take place if an interaction between the bait receptorfusion protein and the prey fusion protein occurs. The prey protein isfused to gp130 which carries STAT binding sites. The receptor-associatedJanus kinases phosphorylate gp130 only after bait-prey interaction,resulting in binding, phosphorylation and subsequent nucleartranslocation of STAT3 transcription factors. STAT-regulated reportergenes are expressed as a function of the bait-prey interaction. Toidentify novel intracellular interaction partners, a selection strategywas established which confers puromycin resistance (Eyckerman, Verhee etal. 2001). Although the method allows detection of a protein-proteininteraction on the membrane by way of expression of a reporter gene, itnevertheless requires at least one interaction partner to couple to saidmembrane receptor. Moreover, the complex quaternary structure of thereceptor-kinase-GP130 multimer is not suitable for analyzing difficultprotein classes such as membrane proteins. Owing to insufficientamplification and stability of the signal, the system is incapable ofanalyzing transient or weak interactions.

Methods based on the transfer of energy quanta of a donor molecule to anacceptor molecule when said molecules are brought into very closeproximity have theoretically few limits. These methods may use variousvariants of the green fluorescent protein (GFP) from Aequorea victoria,which are capable of fluorescence resonance energy transfer (FRET) owingto their specific spectral properties (Siegel, Chan et al. 2000). Asimilar method is based on an energy coupling of the bioluminescence ofthe luciferase-luciferin reaction as energy donor and GFP as energyacceptor. The energy transfer is referred to as bioluminescenceresonance energy transfer (BRET) effect (Xu, Piston et al. 1999).However, the addition of appropriate luciferase substrates is requiredhere. The substantial disadvantages of these methods are the result ofthe sensitivity and difficulty of detection. The detection of FRETeffects in vivo requires both very strong expression and complicatedanalysis. Strong overexpression of proteins in heterologous cells oftenresults in the formation of aggregates, wrong folding or misdirectedsubcellular localization. The method provides no possibility of signalenhancement or signal amplification, a decisive disadvantage which rulesout detection of weak interactions or interactions of weakly expressedproteins. In order to be able to detect a protein interaction ofspectrally compatible GFP fusion proteins by way of FRET effects in thecell, background subtractions and photobleaching analyses must becarried out (Haj, Verveer et al. 2002). Owing to the complex technique,the method is not suitable for high throughput methods of analyzingprotein interactions and, in addition, requires complicated analyses andgreat experience, this being an obstacle to broad application.

All indirect methods previously described have the fundamentaldisadvantage of coupling conversion of the protein interaction to adetection method which requires constant activation or allows inparticular transient interactions to be analyzed only in a very narrowtime window. It is therefore possible only in a very limited way, if atall, to analyze protein interactions in post-mitotic cells or toidentify transient interactions. Automatable detection of interactionsunderlying unknown kinetics is thus not possible.

The methods previously described of analyzing or detectingprotein-protein interactions thus has at least one or more of thefollowing disadvantages:

-   The interactions do not take place in vivo, or at least not in    mammalian cells.-   Large amounts of biological material are required.-   The interactions must be permanent or the analysis must take place    at exactly the right time.-   Measuring the interaction requires complicated measurements and,    respectively, apparatus.-   Detection sensitivity is very limited.-   The analysis of endogenously very weakly expressed genes is    restricted.-   Only binary interactions are detected.-   The rate of false-positive or false-negative interactions is high.-   The analysis of cell type-specifically expressed genes in a complex    tissue assemblage or in cell lines is virtually impossible in    biochemical methods.-   The detection methods are automatable only with difficulty.

SUMMARY OF THE INVENTION

It was the object of the present invention to provide a method ofdetecting and analyzing protein interactions, which overcomes thedisadvantages of the prior art listed above. More specifically, it wasthe object of the present invention to provide a method which also makesweak protein interactions and/or stimulus-induced protein interactionsof a transient nature accessible to analysis by generating in a proteininteraction-dependent manner a permanent detection signal which nolonger depends on analysis in a narrow time window. More specifically,it was also an object of the invention to provide a method of analyzingprotein interactions, which comprises generating a detection signal fordetecting specific protein interactions, which signal is increased, i.e.amplified, compared to classical activation of a reporter gene bytransient transcription activation.

The object of the invention is achieved by providing a method ofdetecting and analyzing protein interactions in a cell, which comprisesthe following steps:

-   a) provision of the activity of at least one enzyme from the group    consisting of recombinases and proteases in the cell as a result of    a protein interaction,-   b) continual generation of an active reporter protein in the cell in    question and, where appropriate, in the daughter cells of said cell,    as a result of the enzymatic activity of step a) for a period of    time which exceeds the duration of the protein interaction of step    a),-   c) generation of a detection signal by the reporter proteins    generated in b).

The novel method overcomes substantial disadvantages of the prior art,since it is particularly suitable for analyzing protein interactions,

-   which are of a transient nature-   which are very weak-   which depend on specific stimuli and take place only in vivo-   which depend on the intrinsic properties of particular eukaryotic    cells-   which depend on a particular cellular state-   which depend on specific modifications-   which depend on a complex topology and environment-   which are coupled to the function of protein complexes.

In the method of the invention, a protein interaction can be detected inany living cells or cell assemblages and may take place completely invivo. Biochemical intervention here is not absolutely necessary.Furthermore, the detection signal increases not gradually, i.e. as afunction of the strength of interaction, but is maximally enhanced byswitching on a permanently strongly expressed reporter gene or byconstant de novo generation of active reporter proteins, even beyond theexistence of said protein interaction.

The analysis can be decoupled in time from the possiblystimulus-dependent and/or transient interaction. This makes possible thesynchronized analysis of a multiplicity of potential interactionpartners, such as, for example, in a screening method. Furthermore, itis possible to study in one experiment sequential stimuli of differentsignal transduction pathways and their possible influence on regulatedprotein interactions, also of unknown partners, or to analyze theinfluence of low molecular weight active compounds on proteininteractions. Detection of the reporters is experimentally easy to carryout and compatible to high throughput methods such as, for example, highthroughput screening methods. Protein interaction-dependent short-timeactivating of the reporter system is already sufficient in order togenerate a permanent, stable and long-term in vivo detectable signal inthe method of the invention. In addition, continual de novo generationof the active reporter protein for a period of time which exceeds theexistence of the protein interaction and the accumulation, possiblyoccurring as a result thereof, of said active reporter protein in thecell increase the signal enormously. The increase here is subject to an“all or nothing principle”, where the detection signal is maximallyactivated, notwithstanding the original strength of interaction, andthis proves an advantage, especially in the case of weak and/orstimulus-induced protein interactions of a transient nature.

Furthermore, spatial separation of the protein interaction from theplace of signal generation in the method of the invention has theadvantage, in comparison with some methods according to the prior art,that the background of false-positive signals should be reduced. Theseproperties are a marked improvement compared to the existing systems.The modularity and enormous flexibility of the system allow the analysesand experiments to be very finely adjusted to the particular problem orto the strength of the interaction required.

The method of the invention of detecting and analyzing proteininteractions in a cell involves providing in step a) the activity of atleast one enzyme from the group consisting of recombinases and proteasesas a result of a specific protein interaction. The activity of theappropriate enzyme in the cell in question may be provided herepreferably either by inducing or increasing expression of said enzyme asa result of said protein interaction, by activating, in particular byproteolytically activating, said enzyme as a result of said proteininteraction, or by transcomplementation of said enzyme as a result ofsaid protein interaction. In the case of transcomplementation of theenzyme, the occurrence of a specific protein interaction results in twofusion proteins, both comprising in each case part of the amino acidsequence of the enzyme (i.e. either of the protease or of therecombinase), coming into spatial proximity to one another and thusbeing able to form a transcomplemented, functional enzyme. Furthermore,the protein interaction-imparted spatial proximity between a proteaseand its substrate may provide the activity of the enzyme.

In step b), the enzyme activity of step a) results in an active reporterprotein being continually generated in the cell in question and, whereappropriate, in the daughter cells of said cell for a period of timewhich exceeds the existence of the protein interaction of step a). Here,the period of time which exceeds the existence of the proteininteraction of step a) is preferably the period which comprises theentire lifetime of the cell in question. More preferably, the activereporter protein is continually generated in the cell in question andadditionally also in the daughter cells of said cell in question oversuch a period of time which comprises the entire lifetime of thedaughter cells. This period which is usually very long and in which theactive reporter protein in the cell in question or additionally even inits daughter cell is continually generated de novo preferably alsoresults in a certain accumulation of said active reporter protein insaid cell in question and/or in its daughter cell.

In method step c), the active reporter protein generated in step b)generates a detection signal which is measured. Accumulation of theactive reporter protein ultimately results in the generation in themethod of the invention of a detection signal which is increasedcompared to a detection signal generated by a reporter protein which hasbeen generated merely during the course of the protein interaction, andwhich therefore has accumulated less. The detection signal may be thefluorescence of a fluorescent reporter protein such as, in particular,GFP and its variants or luciferase. However, the detection signal mayalso be the substrate converted by an enzyme functioning as reporter,such as, in particular, in a β-galactosidase assay. Furthermore,resistance-conferring genes or else genes for growth selection may beused as reporter proteins and the resistances or growth conditionsresulting from expression of said genes may be used as detection signal.

The principle underlying the method is based on a specific proteininteraction converting first to a permanent signal for continual de novogeneration of an active reporter protein in the cell. The signals areactivated by one or more molecular switch systems coupled to one anotherwhich involve proteases and/or DNA double strand-specific recombinases.The system may be designed so as to enable the dynamics of proteininteractions, their coming into being and their dissociation to beanalyzed.

The method of the invention provides in a protein interaction-dependentmanner the activity of at least one enzyme from the group consisting ofrecombinases and proteases in the cell and converts said activity to apermanent detection signal of said cell. The activity of at least oneenzyme from the group consisting of recombinases and proteases initiatesin the cell a switch-like mechanism which ultimately results in theformation of a permanent and, compared to activation of a reporter geneby classical transcription activation, greatly amplified detectionsignal of the cell.

Some preferred embodiments of the method of the invention are based onlyon generation of a permanent signal by providing in a proteininteraction-dependent manner the activity of a recombinase.

These embodiments are based on providing in a proteininteraction-dependent manner a DNA recognition sequence-specificrecombinase and a reporter system coupled to the function thereof.

DNA double strand-specific recombinases of the integrase family maymediate the substitution or integration of various non-homologous DNAmolecules (Lewandoski 2001). The integrase protein family comprises morethan 100 known members of various species (reviewed in (Nunes-Duby, Kwonet al. 1998) and in (Esposito and Scocca 1997)). The molecular switchused in transgenic animals or in cell culture is essentially the Crerecombinase of P1 bacteriophage and yeast recombinase FLP (Sauer 1998)(Buchholz, Angrand et al. 1998). The activity of a cell type-specificrecombinase or a recombinase expressed in a regulated manner duringdevelopment may activate in vivo a downstream reporter gene. This makesit possible to permanently activate various molecular markers which maybe utilized for a multiplicity of analyses in animals (Lewandoski 2001).Cre recombinase was also used in mammalian cells as reporter gene foranalyzing signal transduction mechanisms (Mattheakis, Olivan et al.1999). Furthermore, Cre recombinase-based gene regulation systems havebeen described in which target genes can be inducibly switched on orinactivated and which are based on ligand-controlled nuclear import offusion proteins of Cre recombinase with ligand-binding domains ofvarious nuclear receptors. (Kellendonk, Tronche et al. 1996) (Feil,Brocard et al. 1996) (Metzger, Clifford et al. 1995). DNA doublestrand-specific recombinases which can permanently activate a reportergene in a protein interaction-dependent manner are an ideal molecularswitch system in order to convert transient interactions to permanentsignals. Recombinases and also the great advantages which they provideby generating permanent and, compared to classical transcriptionactivation, greatly amplified detection signals for analyzing proteininteractions have not been described previously in connection with theanalysis of protein interaction.

In the method of the invention, the activity of a recombinase may beexpressed by transcomplementation, by protein interaction-dependenttransport from the cytoplasm into the nucleus or transcriptionfactor-dependent as reporter gene. Transcription of the recombinase maybe induced by in a protein interaction-dependent manner bytranscomplementation, for example by functional reconstitution of atranscription factor, or by protein interaction-dependent transport of afunctional transcription activator from the cytoplasm into the nucleus.

A multiplicity of studies exist on regulation of the activity of Crerecombinase as transcriptional reporter gene or by controlled nuclearimport, both for in vitro and for in vivo applications for activatingdownstream genes (Lewandoski 2001). However, none of these studiesutilizes Cre recombinase as transcriptional reporter gene or by way ofcontrolled nuclear import for analyzing new or known proteininteractions. Likewise no studies about transcomplementation of arecombinase or recombinase activity in connection with the analysis ofprotein interactions have been described. Cre recombinase binds, as aprotein dimer, in each case to a double-stranded DNA recognitionsequence the “loxP” recognition sequence. The active,recombination-mediating complex is formed by a homotetramer and two loxprecognition sequences. Intermolecular transcomplementations of mutantCre proteins, characterized by the loss of different functions, havebeen described (Shaikh and Sadowski 2000). These studies lead to theconclusion that intermolecular transcomplementation of various Cremutants is a possible strategy for analyzing protein interactions.Likewise, an intramolecular transcomplementation strategy seemspossible, owing to functional analyses of chimeric proteins of the FLPand Cre recombinases and, in particular, owing to the known crystalstructure of the protein (Guo, Gopaul et al. 1997). The Cre proteinfolds into two distinct globular domains (AS 20-129 and AS 132-341)which are connected by a short section.

Depending on the application, the following may be downstream reportersystems activated by the recombinase:

-   Directly in vivo and in vitro detectable and quantifiable proteins    (epifluorescent or autofluorescent proteins such as, for example,    green fluorescent protein GFP and its derivatives or Aequorin).-   Indirectly in vivo and in vitro detectable and quantifiable enzymes    (luciferase, beta-galactosidase, alkaline phosphatase,    beta-lactamase, etc.).-   Exposed surface proteins which are biochemically detectable or    suitable for affinity isolation of cells.-   Proteins or enzymes conferring resistance to cytotoxic substances or    minimal media (neomycin/G418, puromycin, blasticidin S, zeocin,    ampicillin, kanamycin, gentamycin, tetracycline, xanthine-guanine    phophoribosyl transferase (XGPT) etc.).-   Cytotoxic or pro-apoptotic proteins (diptheria toxin, activated    caspases, etc.)-   Proteins altering the growth or morphology of the cell in which they    are expressed.

In a preferred embodiment of the method of the invention, step a)provides in a protein interaction-dependent manner the activity of arecombinase by transfecting or infecting the cell with the expressionvector i), said expression vector i) comprising a recombinase gene underthe control of a transcription factor, and continual generation of theactive reporter protein in the cell in question according to step b) iscarried out via the individual steps b1) to b3):

-   b1) transfection or infection of the cell with a construct ii) which    comprises a stop cassette flanked by recombination sites with the    downstream nucleotide sequence of a reporter gene under the control    of a constitutive promoter,-   b2) removal of said stop cassette, flanked by recombination sites,    of construct ii) by means of the recombinase of a),-   b3) constitutive expression of the reporter gene.

The individual transfections or infections of the cell are carried outeither transiently or stably according to standard methods.

If expression of the recombinase is induced in such a cell in a proteininteraction-dependent manner, said recombinase can excise from thenucleotide sequence of the reporter gene the stop cassette flanked bythe recombinase recognition sites. This results in activation of thedownstream reporter gene and thus in permanent expression and constantaccumulation of the functional reporter protein in the cell.

In a further preferred embodiment of the method of the invention,continual generation of the active reporter protein in the cell inquestion according to step b) is carried out via the individual stepsb1) to b3):

-   b1) transfection or infection of the cell with a construct ii) which    comprises a stop cassette flanked by recombination sites with the    downstream nucleotide sequence of a reporter gene under the control    of a constitutive promoter,-   b2) removal of said stop cassette, flanked by recombination sites,    of construct ii) by means of a recombinase provided by protein    interaction-dependent transcomplementation of a functional    recombinase in the nucleus,-   b3) constitutive expression of the reporter gene.

The protein interaction-dependent transcomplementation of a functionalrecombinase in the nucleus according to step b2) is carried out byadditionally expressing heterologously in the cell

-   a first fusion protein comprising at least one first interaction    partner and part of the recombinase, and-   a second fusion protein comprising at least one second interaction    partner and another part of said recombinase.

Accordingly, the occurrence of a specific protein interaction betweentwo interaction partners results in a functional recombinase beingreconstituted in the nucleus, which is then in turn capable of removingthe stop cassette flanked by plox sites or other recombinase-specificrecognition sites from the reporter construct ii), with the reportergene being activated in the process.

A stop cassette in this connection means a DNA insertion which isinserted into the reporter gene in such a manner that its presence leadsto inactivation of said reporter gene. After removing the stop cassetteflanked by the recombination sites by means of the recombinase activityprovided in a protein interaction-dependent manner, nothing now preventsa permanent activation of the reporter gene and thus constantaccumulation of, for example, fluorescent reporter protein or, forexample, enzymatic reporter protein. The accumulation of reporterprotein furthermore results in a considerable increase in the detectionsignal in comparison with reporter gene activation by classicaltranscription activation. The extent of signal amplification here islimited merely by the level of expression of the reporter gene or elseby the turnover of the enzymatic activity of said reporter gene, but notby duration or strength of the protein interaction to be detected.

In addition to the molecular switch system based on the proteininteraction-dependent recombinase activity, the invention also relatesto a further switch system at the molecular level which is based on theactivity of a protease, provided in a protein interaction-dependentmanner, or on coupling of a protease activity with a reporter activatedby proteolytic processing. Similarly to the activity of a recombinase,the short-time activity of a protease may result in generating apermanent detection signal as a result of continual generation of anactive reporter protein in the cell beyond duration of the proteininteraction.

The invention therefore also relates to embodiments of the method of theinvention which utilize both molecular switch systems, one based on arecombinase activity and one based on a protease activity, and toembodiments based only on the activity of one of the molecular switchsystems, either the protease activity or the recombinase activity.

In a preferred embodiment based on providing the activity of a proteaseand of a recombinase, the protease activity is provided bytranscomplementation of a functional protease, in particular of the TEVprotease.

As example 8 shows, transcomplementation of the TEV protease may becarried out by dividing the protease sequence at arbitrary positions ofthe protein, but in particular at the positions between amino acids 60and 80 and at the position between amino acids 95 and 120 of the TEVprotease. The two parts of the TEV protease may also overlap here, aslikewise shown in example 8. Expression of the two TEV proteasefragments to be complemented in each case as fusion protein with apotentially interacting other protein result in fusion proteins compriseprotease fragments each of which per se no longer possesses any proteaseactivity. However, if the fusion proteins, owing to an interaction ofthe two potentially interacting proteins, come into close proximity toone another, the particular protease fragments result in a functionalTEV protease.

According to this example of transcomplementation of the TEV protease,other proteases and also other proteins such as, in particular,recombinases or transcription factors, may also be transcomplemented.The ideal positions for dividing the corresponding protein to betranscomplemented must be tested experimentally in each individual case.

This transcomplementation of the functional protease is carried out herevia the following steps:

-   e) expression of    -   e1) a first fusion protein comprising the first interaction        partner and part of a protease, and    -   e2) a second fusion protein comprising the second interaction        partner and another part of said protease,    -    with, where appropriate, at least one of the two fusion        proteins possessing a further domain which causes said fusion        protein to be anchored outside the nucleus, and    -   e3) expression of a functional recombinase,    -   which, where appropriate, is a further domain of the first or        second fusion protein and can be proteolytically removed from        the other domains via a recognition and cleavage site for the        protease, or expression of a functional recombinase on a third        fusion protein which, in addition to the functional recombinase        itself which is proteolytically removable via a recognition and        cleavage site for the protease, comprises a further domain        causing the third fusion protein to be anchored outside the        nucleus, in a cell,-   f) reconstitution of a functional protease due to the first and    second interaction partners interacting with one another,-   g) proteolytic removal of the functional recombinase from its    anchoring position outside the nucleus by the reconstituted protease    of f),-   h) transport of the functional recombinase into the nucleus and    activation of a recombinase-dependent reporter gene.

The component e3) here possesses preferably a domain for its anchoringoutside the nucleus, which results in anchoring on the cell membrane.

In addition, the cytoplasmic structure outside the nucleus, on which atleast one of the two fusion proteins is anchored via a domain suitabletherefor, may, however, also be any other membrane-enclosed cellorganelle, with the exception of the nucleus. Particular proteolyticallyremovable protein targeting domains which effect targeting of thecarrier protein into the lumen of a particular cell organelle may beused for fixing at least one of the two fusion proteins outside thenucleus.

In a further, likewise preferred embodiment the activity of a proteaseis provided in a protein interaction-dependent manner by generatingspatial proximity between a functional protease and its substrate by thefollowing steps:

-   j) expression of    -   j1) a first fusion protein comprising the first interaction        partner and a functional protease, and    -   j2) a second fusion protein comprising the second interaction        partner, a functional recombinase domain and a further domain        causing anchoring outside the nucleus, with at least said        functional recombinase domain being proteolytically removable        from the domain which causes the second fusion protein to be        anchored outside the nucleus, via a recognition and cleavage        site of the protease used, the cell,-   k) effecting a spatial proximity, resulting from the interaction of    the first and the second interaction partner, between the functional    protease of the first fusion protein and the protease recognition    and cleavage site on the second fusion protein,-   l) proteolytically removing the functional recombinase anchored    outside the nucleus by cleaving the protease cleavage site with the    proximal protease, transporting the free functional recombinase into    the nucleus and activating a reporter system.

In another preferred embodiment of the method of the invention, theactive reporter protein of step b) is continually generated in the cellin question by providing, as a direct or indirect result of the proteininteraction, a specific functional transcription factor in the nucleusof said cell, which induces or increases expression of said reporterprotein.

This protein interaction-dependent provision of a specific functionaltranscription factor in the nucleus of the cell may be accomplished herepreferably by the protein interaction-dependent transcomplementation ofa functional transcription factor in the nucleus.

Said protein interaction-dependent transcomplementation of a functionaltranscription factor in the nucleus may be carried out, in particular,via the following individual steps:

-   -   m) expression of a first fusion protein comprising the first        interaction partner and part of the transcription factor and of        a second fusion protein comprising the second interaction        partner and another part of the transcription factor,    -   n) reconstitution of a functional transcription factor due to        said first and second interaction partner interacting with one        another,    -   o) induction of expression of a functional recombinase for        activation of a recombinase-dependent reporter system in the        nucleus.

In another preferred embodiment, the specific functional transcriptionfactor which induces or increases expression of the reporter protein maybe provided by the protein interaction-mediated spatial proximitybetween a protease and its substrate.

Said spatial proximity between a protease and its substrate in thenucleus is preferably generated here by the following steps:

-   j) expression of    -   j1) a first fusion protein comprising the first interaction        partner and a functional protease, and    -   j2) a second fusion protein comprising the second interaction        partner, a functional transcription factor domain and a further        domain causing anchoring outside the nucleus, with at least said        functional transcription factor domain being proteolytically        removable from the domain which causes the second fusion protein        to be anchored outside the nucleus, via a recognition and        cleavage site of the protease used, in the cell,-   k) effecting a spatial proximity, resulting from the interaction of    the first and the second interaction partner, between the functional    protease of the first fusion protein and the protease recognition    and cleavage site on the second fusion protein,-   l) proteolytically removing the functional transcription factor    anchored outside the nucleus by cleaving the protease cleavage site    with the proximal protease, transporting the free functional    transcription factor into the nucleus and activating a reporter    system.

In a further preferred embodiment of the method of the invention,protein interaction-dependent transcomplementation of a proteaseprovides the specific functional transcription factor in the nucleus ofthe cell.

This protein interaction-dependent transcomplementation of the proteasemay be achieved, in particular, by the following steps:

-   p) expression of    -   p1) a first fusion protein comprising the first interaction        partner and part of a protease, and    -   p2) a second fusion protein comprising the second interaction        partner and another part of said protease,-   with, where appropriate, at least one of the two fusion proteins    possessing a further domain which causes the fusion protein to be    anchored outside the nucleus, and    -   p3) expression of a functional transcription factor, which,        where appropriate, is a further domain of the first or second        fusion protein and which is proteolytically removable from the        other domains of said fusion protein via a recognition and        cleavage site for a protease, or expression of a functional        transcription factor on a third fusion protein which, in        addition to the functional transcription factor itself which is        proteolytically removable via a recognition and cleavage site        for said protease, comprises a further domain which causes said        third fusion protein to be anchored outside the nucleus;    -    in a cell;-   q) reconstitution of a functional protease due to the first and    second interaction partners interacting with one another;-   r) proteolytically removing the functional transcription factor from    its anchoring position outside the nucleus by the reconstituted    protease of q)-   s) providing a functional transcription factor in the nucleus and    subsequently inducing expression of a recombinase-dependent or a    recombinase-independent classical reporter system.

In a further preferred modification of the method, the functionaltranscription factor proteolytically removed in step r),

-   -   induces expression of a recombinase-independent, classical        reporter system    -   and additionally induces expression of the gene of a functional        protease for further continual activation of the reporter system        employed in the nucleus is step s).

The additional transcriptional activation of the gene of a functionalprotease by the activity of the functional transcription factor hereresults in continual generation of an active reporter protein withoutthe use of a recombinase activity.

Another modified embodiment relates to a method of detecting andanalyzing protein interactions in a cell, which comprises the followingsteps:

-   u) expression of    -   u1) a first fusion protein comprising the first interaction        partner and part of a protease, and    -   u2) a second fusion protein comprising the second interaction        partner and another part of said protease, and    -   u3) a reporter which can be activated or inactivated by        proteolysis, in the cell,-   v) reconstitution of a functional protease due to said first and    second interaction partner interacting with one another,-   w) activation of the proteolysis-activatable or inactivation of the    proteolysis-inactivatable reporter by the reconstituted functional    protease of step v).

In this embodiment, the component u3) expressed in step u) is preferablya proteolysis-activatable reporter protein whose reporter activity isinactivated by insertion of an additional amino acid sequence and/or byinsertion of at least one recognition and cleavage site for a proteaseand can be proteolytically activated by the activity of a protease.

As an alternative to this, however, it is also possible for thecomponent u3) expressed in step u) to be a proteolysis-inactivatablereporter protein which contains at least one recognition and cleavagesite for a protease and whose reporter activity can be proteolyticallyinactivated.

In this embodiment, providing a protease activity bytranscomplementation of a functional protease may alternatively also beprovided by the protein interaction-mediated spatial proximity betweensaid protease and its substrate. In this case, the individual methodsteps of this embodiment are modified as follows.

-   x) expression of    -   x1) a first fusion protein comprising the first interaction        partner and a functional protease, and    -   x2) a second fusion protein comprising the second interaction        partner and a reporter which can be activated or inactivated by        proteolysis, in the cell;-   y) interaction of said first and second interaction partners with    one another;-   z) activation of the proteolysis-activatable reporter or    inactivation of the proteolysis-inactivatable reporter by protein    interaction-dependent spatial proximity of the functional protease    of the first fusion protein x1) and the proteolysis-activatable or    inactivatable reporter of the second fusion protein x2).

The invention furthermore relates to a method of detecting and analyzingprotein interactions in a cell, which comprises the following steps:

-   J) expression of    -   J1) a first fusion protein comprising the first interaction        partner and part of a protease, and    -   J2) a second fusion protein comprising the second interaction        partner and another part of said protease, and    -   J3) constitutive expression of a reporter protein which is        anchored via a suitable domain outside the nucleus and which can        be proteolytically removed from said anchoring position, and        additionally comprises a further domain which, after proteolytic        removal, effects localization of said reporter protein into a        particular compartment of the cell,    -    in the cell,-   K) reconstitution of a functional protease due to said first and    second interaction partners interacting with one another,-   L) proteolytically removing the reporter protein together with its    domain which causes localization of the free reporter protein into a    particular compartment of the cell, by the functional protease of    step K),-   M) detecting the altered location of the reporter protein.

Detection systems based on the proteolytic removal of membrane-boundtranscription factors have already been developed for application inyeast to isolate new proteases or protease-regulating proteins ormolecules (Kamada, Kusano et al. 1998) (Hirowatari, Hijikata et al.1995) (Broad 1999). The use of exogenous proteases in connection withthe analysis of protein interactions has not been described previously.

A proteolytic activity of this kind may take place, as described indetail above, by transcomplementation or by spatially bringing together(proximity) the enzyme and its substrate in a proteininteraction-dependent manner. Depending on the application, thefollowing proteins or enzymes may be activated by proteolytic cleavage:

-   A recombinase bound outside the nucleus, which enters the nucleus    and can become active there only after proteolytic removal.-   A transcription factor fixed outside the nucleus which enters the    nucleus and can become active there only after proteolytic removal.-   A GFP variant fixed outside the nucleus which enters the nucleus    only after proteolytic removal. Detection is carried out by way of    the altered morphology of the fluorescence signal.-   An enzyme or protein or molecule or protein pair or molecule pair    activatable or inactivatable by proteolytic cleavage. These may be    recombinases, proteases, GFP variants, enzymes or cellular signal    proteins.-   A modified protease which likewise can become active only after    proteolytic cleavage. The latter results in permanent and maximum    signal activation due to a cascade of successive proteolytic    cleavages.-   A transcription activator whose nuclear localization signal is    masked by a protein domain which is located on the same or on    another protein and contains a protease recognition site so as for    the nuclear localization signal of said transcription activator to    be recognized only after proteolytic cleavage and the latter to be    able to enter the nucleus only then.

An essential feature of the present invention is the fact that proteininteractions in the cell are recorded at those places where they alsonaturally occur, for example in the case of ER residence proteins, inthe ER or, in the case of surface receptors, on the plasma membrane. Thetype of anchoring and the localization resulting therefrom of aproteolytically removable transcription activator, a recombinase oranother protease sensor such as, for example, a proteolyticallyactivatable or inactivatable enzyme or a proteolytically activatable orinactivatable fluorescent protein make it possible to specificallydetect protein interactions at their natural location within the celland, in addition, to investigate at which locations within the cell saidinteraction takes place. The method of the invention therefore comprisesvarious methods of specific anchoring or localization of theabovementioned protease sensors. The proteolytically removable proteinswhich display their activity in the nucleus, such as transcriptionactivators or transcription factors or recombinases, must be anchored insuch a way that they are located on the cytoplasmic side of membranes orcell compartments in order to be able to enter the nucleus afterproteolytic removal from the anchoring position. Depending on theapplication, this involves the following anchorings, for example:

-   -   For localizations on the ER membrane, fusion of the        proteolytically removable transcription activator or of the        recombinase or of a protease sensor (summarized as proteins to        be anchored) to the C terminus of a type I or type III membrane        protein with ER retention signal, which resides in the ER, with        a protease cleavage site being located between the anchoring        protein and the anchored protein.    -   For localizations in the Golgi apparatus, fusion to        Golgi-residing membrane proteins or, alternatively,        isoprenylation of the protein to be anchored or of the protease        sensor by appending a protease cleavage site followed by a        geranylgeranylization signal, e.g. CVIL, or a farnesylization        signal, e.g. CIIM, to the C terminus.    -   For localizations on the plasma membrane, fusion of the protein        to be anchored to the C terminus of a type I or type III        membrane protein or to the C terminus of the transmembrane        region of such a membrane protein, with a protease cleavage site        being located between the anchoring protein and the anchored        protein.    -   For localizations on the plasma membrane, fusion of the protein        to be anchored to the N terminus of a type II membrane protein        or to the N terminus of the transmembrane region of such a        membrane protein, with a protease cleavage site being located        between the anchoring protein and the anchored protein.

Further, for localizations on the plasma membrane, fusion with proteindomains carrying lipid modifications such as myristoylation orpalmitoylation, with a protease cleavage site being located between theanchoring protein and the anchored protein.

For localizations on peroxysomes or mitochondria, fusion to a membraneprotein located in the peroxysomal membrane or the outer mitochondrialmembrane in such a way that the anchored protein is located on thecytoplasmic side and can be removed via a protease cleavage site.

Suitable for analyzing the interaction of cytoplasmic proteins areanchorings of the proteolytically removable transcription activator orof the recombinase or of a protease sensor on the cycoskeleton of thecell, for example by fusion to actin.

Protease sensors capable of directly generating a measurable signal,independently of their localization, such as proteolytically activatableor inactivatable fluorescent proteins or enzymes, may, in addition tothe methods mentioned above, also be anchored by appropriate proteinfusions on the inside of organelle membranes or be present in a solubleform in the lumen of said organelles due to organelle-specific signalsequences. Examples of such signal sequences are the peroxysome-specificsignal, peroxysomal targeting signal 1 (PTS1, SKL), on the C terminus ofproteins or N-terminal mitochondrial targeting sequences such as, forexample, the first 31 amino acids of the prepeptide of cytochrome Coxidase subunit VIII.

Proteolytic enzymes which are particularly suitable for intracellularuse are the potyvirus NIa proteases such as, in particular, the 27 kDaNIa protease of the Tobacco Etch Virus (referred to as “TEV protease”hereinbelow). The TEV protease is very well tolerated in eukaryoticcells and exhibits specific activity in the cytosol (Faber, Kram et al.2001) (Uhlmann, Wernic et al. 2000). The TEV protease is a member of theC4 family of cysteine proteases, and the primary structure isdistinguished by the characteristic distribution of the amino acids ofthe catalytic triade, histidine (position 46), aspartate (position 81)and cysteine (position 151), (Ryan and Flint 1997). Thethree-dimensional structure, in contrast, is little known but asecondary structure prediction and comparison with other proteases whose3D structure has already been resolved implicate a large homology to thetrypsin-like serine proteases (Bazan and Fletterick 1988) (Bazan andFletterick 1989). Their characteristic structural feature is the highproportion of B-sheet domains in the secondary structure which fold togive a typical bilobal overall structure. In the process, the catalyticamino acids histidine and arginine are distributed to the N-terminallobe and the serine (or cysteine) is located on the C-terminal lobe.This distribution of the three catalytic amino acids to the two“hemispheres” of the protease serves as the basis for thetranscomplementation strategy. Several variants are conceivable herewhich involve dividing the protein into two fragments on which then ineach case one or two of the amino acids of the triade can be found. Theindependent folding of said fragments is crucial for functionaltranscomplementation. In order to ensure this folding, it may benecessary to generate overlapping fragments and to test these foractivity. The aim of transcomplementation is to choose the fragments insuch a way that they possess per se no activity and regain this activityonly when being fused to interacting proteins, interacting proteindomains or other interacting molecules.

Owing to their large structural homology (alignment in (Barrett-A J,Rawlings-N D et al. 1998)), all proteases are suitable in principle forthe method. It is required, however, that their presence in thecorresponding cells or cell compartments is tolerated. Within theframework of the invention, the protease renin which is usually secretedinto the blood stream was also expressed in PC-12 cells. It was possibleto detect the intracellular activity with a reporter gene constructequipped with the specific recognition site from the renin substrateangiotensinogen renin is theoretically very well suited to atranscomplementation strategy, since this protease is very homologous toHIV protease 1 which in turn is known for the functional molecule beingcomposed of two identical subunits.

In addition, it is also possible to carry out transcomplementation of aprotease on the cell surface or extracellularly. For this purpose, thefragments and possibly reporters would have to be fused to membraneproteins, proteins of the extracellular matrix or secreted proteins insuch a way that they project into the medium outside the cell or aresecreted. In such a version of the method, the activity may be detectedby adding a substrate or by coexpressing an appropriately modifiedreporter. Conversion of a specific substrate (e.g. by fluorescentlycoupled substrate peptides or enzymes whose activity is destroyed byspecific proteolysis) ultimately allows transcomplementation to beanalyzed in a completely cell-free manner by studying recombinantly orin-vitro produced fusion proteins in the reaction vessel.

The protein interaction-dependent activity of a recombinase and/oractivity of a protease in the above-described embodiment of theinvention directly or indirectly leads intracellularly to permanentactivation of corresponding reporter genes or reporter proteins in thecell. A combination of both systems (the recombinase-based and theprotease-based switch systems) allows high sensitivity and provides manypossibilities for finely regulating the detection limit or thesignal-to-background ratio.

The molecular switch systems are based either on a recombinase activityor a protease activity, or a combination of both systems may alsoinvolve molecular feedback mechanisms which ultimately result in avirtually endless activation of the reporter.

A preferred embodiment of the method of the invention, which comprisessuch a molecular feedback mechanism for virtually endless signalincrease, includes the following individual steps:

-   A) expression of    -   A1) a first fusion protein comprising the first interaction        partner and part of a protease, and    -   A2) a second fusion protein comprising the second interaction        partner and another part of said protease, and    -   A3) a protease which can be activated by proteolysis or        inactivated by proteolysis,    -   in the cell,-   B) transcomplementation of a functional protease by the first and    second interaction partners interacting with one another,-   C) activation of the proteolytically activatable proteases by the    transcomplemented functional protease of step B),-   D) activation of a proteolytically activatable or a proteolytically    inactivatable reporter system by the functional proteases of    steps B) and C).

In this embodiment, the occurrence of a specific protein interactionfirstly causes transcomplementation of the protease. The functionalprotease transcomplemented due to protein interaction may then activateby proteolysis both a protease which is constitutively expressed in thecell and which itself is proteolytically activatable and the likewiseproteolytically activatable, constitutively expressed reporter protein.Finally, the, similarly to a chain reaction, increasing number offunctional protease molecules activated by proteolysis may result in theproteolytically activatable, likewise constitutively expressed reporterprotein being provided permanently and virtually endlessly.Alternatively, it is also possible in this embodiment for aproteolysis-inactivatable reporter protein to be constitutivelyexpressed in the cell.

The classical reporter genes which in this embodiment are expressed inthe cell in a proteolytically activatable or proteolyticallyinactivatable form include in this connection the following reporters:

-   Directly in vivo and in vitro detectable and quantifiable proteins    (epifluorescent or autofluorescent proteins such as, for example,    green fluorescent protein GFP and its derivatives or Aequorin).-   Indirectly in vivo and in vitro detectable and quantifiable enzymes    (luciferase, beta-galactosidase, alkaline phosphatase,    beta-lactamase, and the like).-   Exposed surface proteins which are biochemically detectable or    suitable for affinity isolation of cells.-   Proteins or enzymes conferring resistance to cytotoxic substances or    minimal media (neomycin, puromycin, blasticidin S, zeocin,    ampicillin, kanamycin, gentamycin, tetracycline, xanthine-guanine    phosphoribosyl transferase (XGPT) and the like).-   Cytotoxic or pro-apoptotic proteins (diptheria toxin, activated    caspases, and the like).-   Proteins altering the growth or morphology of the cell in which they    are expressed.

In a modification, the interaction may be detected locally in thenucleus independently of the described reporter system by usingalternative reporters. A protease transcomplemented due to proteininteraction activates a proteolytically activatable protease and aproteolysis-activatable reporter protein such as GFP, for example.Immediately after synthesis, the constitutively expressed components,the proteolytically activatable protease and the proteolyticallyactivatable reporter protein, are cleaved again, resulting in continualactivation. The detection may take place in principle in eachcompartment and outside the cell. After fusion to appropriate signalsequences, the components are sorted into the corresponding compartmentsand are processed and activated there. The detection is carried out insitu.

Further modifications of the method above of analyzing proteininteractions with virtually endless signal increase are the followingembodiments 1.) and 2.) which are discussed in more detail below:

-   1. In this modification, a protease transcomplemented due to a    specific protein interaction proteolytically activates a    transcription factor which then, in addition to a conventional    reporter gene such as, for example, beta-galactosidase, luciferase,    GFP variants, etc. also switches on expression of an intact version    of the same protease. Activation of the components is thus followed    by continual activation of the complete reporter system: immediately    after its synthesis, the constitutively expressed proteolytically    activatable transcription factor is independently cleaved again by    another protease activity provided by transcomplementation, since    the protease coexpressed with the reporter gene is permanently    available and can accumulate in the cell.-   2. In this further modification, a protease transcomplemented due to    protein interaction activates a proteolytically activatable protease    and a proteolysis-activatable reporter protein such as, for example,    GFP or its variants. The proteolytic activatability of the reporter    protein and of the protease, respectively, may be achieved, for    example, by inserting one or more recognition sites for the protease    into the corresponding protein at the DNA level. After their    synthesis, the constitutively expressed components, the    proteolytically activatable protease and the proteolytically    activatable reporter protein, are proteolytically cleaved and thus    activated. In this way there are subsequently always sufficiently    functional proteases available which in turn can activate    continuously the constitutively expressed proteolytically    activatable reporter proteins. The detection signal thus increases    itself and is thus permanent.    -   Detection may take place in principle also in any compartment of        the cell and also outside the cell. After fusion with        appropriate signal sequences, the components are sorted here        into the corresponding compartments and processed and activated        there. Detection is carried out here in situ.-   3. In this modification of the method, a reporter protein which can    be activated or inactivated by proteolysis is constitutively    expressed in the cell and is then proteolytically cleaved by protein    interaction-dependent transcomplementation of a protease activity    and thus, depending on the design of the system, either activated or    inactivated. In this embodiment too, the detection signal is    increased to a certain extent, since the constitutively expressed    proteolytically activatable or inactivatable reporter protein can    accumulate in the cell and is thus present in excess. The signal    strength may be further increased by using as reporter gene, for    example, enzymes with quantifiable activity and with a high turnover    rate.-   4. In this modification, which is very closely related to the    embodiment 3.), a first fusion protein comprising the first    interaction partner and a functional protease, and a second fusion    protein comprising the second interaction partner and a reporter    which can be activated or inactivated by proteolysis    -   are heterologously expressed in the cell.    -   If a specific protein-protein interaction takes place between        the first and the second interaction partner, a close spatial        proximity is formed between the functional protease and the        reporter protein activatable or inactivatable by proteolysis.        Proteolytic activation or inactivation of the reporter protein        then, like in embodiment 3.), results in an        interaction-dependent detection signal.

The invention furthermore relates to screening methods for identifying aspecific interaction partner of a bait protein by carrying out any ofthe mentioned methods of the invention. Particular preference is givenhere to screening methods which make use of a cDNA library or an “ORF”(open reading frame) library.

The inventive methods of detecting protein interactions also permitdetection of the dissociation of specific protein-protein interactions.Here, the protease activity or recombinase activity is functionallycoupled directly to the protein-protein interaction, this being done insuch a way that the occurrence of said protein-protein interactioninitially does not effect any activation of said protease or recombinaseand therefore does not yet cause any permanent activation of thereporter system used. Only the active dissociation then results inactivation of the proteases or recombinases and subsequently inactivation of the downstream reporter system.

All of the above-described inventive embodiments of analyzing anddetecting associations between two interacting proteins may thereforealso be used in their “reverse” embodiment. The present inventiontherefore also relates expressly to these “reverse” embodiments whichlikewise comprise the generation of permanent detection signals whichare increased compared to the classical transcription activation ofreporter systems. In some embodiments, even virtually endlesslyincreased detection signals are generated which indicate the induceddissociation of a specific interaction.

The “reverse” embodiments are especially suitable for those problemsconcerning the analysis of the dynamics of a specific protein-proteininteraction involving two known interaction partners. They are moreoversuitable for determining substances having an influence on the constancyof said interaction, thus in particular inhibitors or else activators ofsaid interaction. Thus it would be possible, in particular, to carry outa high throughput screening method, in particular using a library of lowmolecular weight substances, in order to identify a specific, possiblytherapeutically usable inhibitor of a physiologically important proteininteraction, which may also be relevant to diseases.

The reverse embodiments of the method of the invention accordinglycomprise providing a recombinase activity or protease activity in thecell as a result of the induced dissociation of a defined interactionbetween interaction partners, in particular between proteins or proteincomplexes, which are ultimately converted in the cell to a permanentdetection signal, i.e. which results, in particular, in activation of arecombinase-dependent reporter system or in activation of a classicalreporter system, where appropriate with coexpression of a functionalprotease, for permanent activation of the complete reporter system.

Accordingly, the invention also relates to a method of detecting andanalyzing the dissociation of a defined protein interaction in a cell,which method comprises the steps

-   P) provision of the activity of at least one enzyme from the group    consisting of recombinases and proteases in the cell as a result of    the dissociation of a protein interaction,-   Q) continual generation of an active reporter protein in the cell in    question or, where appropriate, additionally in the daughter cells,    as a result of the enzyme activity of step P) for a period of time    which exceeds the duration of the dissociated state of the protein    interaction,-   R) generation of a detection signal by the reporter proteins    generated in Q).

In this method too, preference is given to continually generating theactive reporter protein in step Q) for such a period of time whichcomprises the entire lifetime of the cell in question.

This method involves generating the active reporter protein in step Q)in particular not only in the cells in question themselves, butadditionally also in the daughter cells of the cell in question, so asto comprise the entire lifetime of said daughter cells.

In all reverse embodiments of the method of the invention, preference isgiven to detecting stimulus-induced dissociations of a transient natureof the interaction partners in question, since the continual generationof an active reporter protein in this method for a period of time whichexceeds the duration over the duration of the dissociated state of theprotein interaction, and the accumulation of said reporter protein,resulting therefrom, and also the increased detection signal generatedthereby entail particular advantages for dissociations of a transientnature.

The induced dissociation of the defined interaction between proteins orprotein complexes is preferably caused by the presence of a specificinhibitor of a protein interaction or by the presence of a specificstimulus which influences the stability or lifetime of a proteininteraction.

The classical reporter systems include within the scope of the presentinvention fluorescent reporter proteins such as GFP and all itscommercially available variants, enzymes with detectable activity, suchas luciferase, beta-galactosidase, etc., or genes conferring resistanceor genes whose expression is required for the cells to grow underparticular deficiency conditions. It is furthermore possible to use inthe embodiments of the invention all proteolytically activatable andproteolytically inactivatable forms of the abovementioned reporter genesystems.

In a preferred reverse embodiment of the method of the invention, stepsa) and b), as a result of the induced dissociation of a proteininteraction, are carried out via the following individual steps:

-   S) expression and interaction of    -   S1) a first fusion protein comprising the first interaction        partner and a functional recombinase, and    -   S2) a second fusion protein comprising the second interaction        partner and an inhibitor of said recombinase, in the cell,-   T) induced dissociation of the interacting fusion proteins, thereby    removing the proximity between the recombinase and its inhibitor,-   U) activation of a recombinase-dependent reporter system by the    functional recombinase of step T).

This reverse embodiment of the method may also be modified in such a waythat, as a result of the dissociation of a defined protein interaction,the activity of a recombinase is provided and that continual generationof the active reporter protein according to step b) is carried out byproviding, as a function of said dissociation of said defined proteininteraction, a specific transcription factor in the nucleus of the cell,which then induces expression of a recombinase and, as a result of this,activates a recombinase-dependent reporter gene.

In detail, this embodiment may preferably be accomplished via thefollowing individual steps:

-   V) expression and specific interaction of    -   V1) a first fusion protein comprising the first interaction        partner and a functional protease, and    -   V2) a second fusion protein comprising the second interaction        partner and an inhibitor for said functional protease, and    -    with, where appropriate, at least one of the two fusion        proteins possessing a further domain resulting in the anchoring        of the fusion protein outside the nucleus, and    -   V3) expression of a functional protein selected from the group        consisting of transcription factors, recombinases and        proteolytically activatable or inactivatable reporter proteins,    -    which protein, where appropriate, is a further domain of the        first or the second fusion protein and which is proteolytically        removable from the remaining domains by a recognition and        cleavage site for the protease,    -   or which, where appropriate, is a further constitutively        expressed fusion protein and a domain for anchoring outside the        nucleus and which is proteolytically removable from its        anchoring position via a recognition and cleavage site for said        protease,    -   V4) where appropriate, expression of a proteolytically        activatable protease, in the cell,-   W) induced dissociation of the interacting fusion proteins, thereby    removing the proximity between the protease and its inhibitor and    providing a functional protease,-   X) proteolytically removing the functional recombinase or the    functional transcription factor of R3) from its anchoring position    outside the nucleus by the functional protease of step W) and    transport into the nucleus,-   Y) activation of a recombinase-dependent reporter system or    -    proteolytic activation of the proteolytically activatable        protease of Rd) by the functional protease of step W),-   Z) activation or inactivation of the proteolytically activatable or    inactivatable reporter proteins of R3) by the functional proteases    of step W) and of step Y).

The fusion of a functional protease to an interaction partner A andfusion of a specific protease-inhibiting protein or peptide tointeraction partner B block the proteolytic activity. Said interactionpartners may also be components of a protein complex. Afterdissociation, the protease becomes active and can activate the reportersystem. Coupling to a permanently activated reporter system may alsodetect transient dissociation.

A constitutively expressed functional protein component which isinitially anchored outside the nucleus and which is particularlysuitable here, is a transcription factor suitable for activating areporter gene or else a recombinase whose activity induces, by excisinga stop cassette flanked by recombination recognition sites from areporter gene, expression of said reporter gene.

Accordingly, in a modification of the reverse embodiment above, thefunctional protein V3) expressed in step V) is a functionaltranscription factor proteolytically removable from its anchoringposition outside the nucleus and which is proteolytically removed fromits anchoring position in step X) by the functional protease of step W)and which activates in step Y) a recombinase-independent, classicalreporter gene.

In another modification of the reverse embodiment above, the furtherfunctional protein V3) expressed in step V) is thus a functionalrecombinase proteolytically removable from its anchoring positionoutside the nucleus and which in step X) is proteolytically removed fromits anchoring position by the functional protease of step W) and whichactivates in step Y) a recombinase-dependent reporter gene.

Alternatively to expressing a functional protein component anchoredoutside the nucleus it is also possible to express heterologously aproteolytically activatable or proteolytically inactivatable reporterprotein in the cell, which can then be proteolytically activated orinactivated by the protease activity provided in adissociation-dependent manner. This may be converted into a permanentdetection signal by coexpressing a proteolytically activatable protease.

In a further modification of the reverse embodiment above, the furtherfunctional protein V3 expressed in step V) is a proteolyticallyactivatable reporter protein or a proteolytically inactivatable reporterprotein which is proteolytically activated or proteolyticallyinactivated in step X) by the functional protease of step W) and whichis directly quantified, with step Y) being dispensed with. Theproteolytically activatable or proteolytically inactivatable reporterprotein need not necessarily be anchored outside the nucleus here.

In a further reverse embodiment of the method of the invention and as aresult of the induced dissociation of a specific protein interaction,the activity of a recombinase is provided and converted to a permanentdetection signal by the following steps:

-   -   expression and specific interaction of    -   a first fusion protein comprising the first interaction partner        and a transcription factor having a DNA binding domain and a        weak transcription activation domain, and    -   a second fusion protein comprising the second interaction        partner and a strong transcriptional repression domain,        in the cell,    -   induced dissociation of the interacting fusion proteins,    -   induction of transcription of the recombinase gene by the first        fusion protein.

In this method, the transcription factor which is functional per se andwhich is contained in the first fusion protein is kept inactive by thefunctionally dominating strong transcriptional repression domain on thesecond fusion protein, until an induced dissociation of the specificprotein-protein interaction takes place and thus the proximity betweenthe transcriptional repression domain and the transcription factor isremoved. After dissociation of the interacting components, the reportergenes are thus expressed and permanently activate the reporter system.

In a further reverse modification of the method of the invention, theinteraction of two fusion proteins may mask a transport signal (e.g. anuclear import signal of one of the partners). This transport signal isreleased again only after dissociation of the protein interaction, andthe corresponding fusion protein can only then enter the nucleus and, asactivating transcription factor, activate reporter gene systems.

The invention furthermore relates to a screening method for identifyingand characterizing specific inhibitors of a defined protein interactionor for identifying and characterizing a defined stimulus whichinfluences a defined protein interaction by carrying out any of thereverse methods of the invention mentioned above.

The invention furthermore relates to the cells into which the proteincomponents required in the various embodiments of the invention havebeen heterologously incorporated at the DNA level in expression vectors.To this end, the cells may be incorporated either stably or transientlywith the appropriate expression vectors comprising expression cassettesof the desired protein components.

The invention thus relates to to a cell which has been transfected orinfected with at least one expression vector, said expression vectorcomprising at least one, preferably at least two, in particular at leastthree, of the constructs i) to vii).

The constructs i) to vii) are defined here as follows:

-   i) a construct comprising a recombinase gene being under the control    of a transcription factor-   ii) a construct comprising a stop cassette flanked by recombination    sites with the downstream nucleotide sequence of a reporter gene    under control of a constitutive promoter,-   iii) a construct comprising a recombinase which is anchored outside    the nucleus and which can be proteolytically removed,-   iv) a construct comprising a transcription factor which is anchored    outside the nucleus and which can be proteolytically removed,-   v) a construct comprising a proteolytically activatable or    inactivatable reporter protein,-   vi) a construct comprising a proteolytically activatable protease,-   vii) a protease gene under the control of a transcription factor.

The cells used for the method of the invention are either yeast cells,bacterial cells or cells of higher eukaryotic organisms (in particularneuronal cells and mammalian cell lines [e.g. embryonic carcinoma cells,embryonic stem cells, P19, F9, PC12, HEK293, HeLa, Cos, BHK, CHO, NT2,SHSY5Y cells]).

The invention further relates to a kit for detecting and analyzingprotein interactions in a cell, which comprises expression vectorscomprising the nucleic acid constructs 1a) and 2a) in each case underthe transcriptional control of a heterologous promoter:

-   1a) a first nucleic acid construct coding for a first fusion protein    comprising the nucleic acid sequence coding for a first protease    fragment and a cloning site suitable for cloning the bait protein in    the reading frame of the first protease fragment, and-   2a) a second nucleic acid construct coding for a second fusion    protein comprising the nucleic acid sequence coding for a second    protease fragment and a cloning site suitable for cloning the prey    protein in the reading frame of the second protease fragment,    and which, where appropriate, comprises expression vectors    comprising at least one of the following constructs:-   3a) a nucleic acid construct coding for a functional recombinase or    for a functional transcription factor which can be proteolytically    removed via a recognition and cleavage site for a protease from a    domain for anchoring to a cytoplasmic structure, it being possible    for said components to be used either as further moieties of the    first or second fusion protein or as separate third fusion protein;-   4a) a nucleic acid construct coding for a recombinase under the    control of the functional transcription factor of No. 3;-   5a) a nucleic acid construct comprising a stop cassette flanked by    recognition sites for recombinases with the downstream nucleotide    sequence of a reporter gene under the control of a constitutive    promoter;-   6a) a nucleic acid construct coding for a proteolytically    activatable or proteolytically inactivatable reporter protein under    the control of a promoter;-   7a) a nucleic acid construct coding for a proteolytically    activatable protease under the control of a promoter.

The kit may also contain expression vectors which, instead of theabovementioned nucleic acid constructs 1a) and 2a), also comprisemodified variants of these two constructs, namely a first nucleic acidconstruct which comprises a functional protease and a cloning site forcloning the bait protein in the reading frame of said protease and asecond nucleic acid construct which comprises a functional,proteolytically removable protein from the group consisting ofrecombinases or transcription factors and a cloning site for cloning theprey protein in the reading frame of said removal protein.

The kit may furthermore also contain expression vectors which, insteadof the nucleic acid constructs 1a) and 2a) above, also comprise furthermodified variants of these two constructs, namely in particular a firstnucleic acid construct which comprises a functional enzyme from thegroup consisting of proteases and recombinases and a cloning site forcloning the bait protein in the reading frame and a second nucleic acidconstruct which comprises the sequence for a functional inhibitor oractivator for the functional enzyme from the group consisting ofproteases and recombinases and a cloning site for cloning the preyprotein in the reading frame.

The expression vectors are preferably provided in the kit in such a waythat the individual components of a cDNA library have already beencloned into the cloning site of the second nucleic acid construct.

The invention further relates to a kit for detecting and analyzingprotein interactions in a cell, which comprises expression vectorscomprising the nucleic acid constructs 1b) and 2b) in each case underthe transcriptional control of a heterologous promoter:

-   1b) a first nucleic acid construct coding for a first fusion protein    comprising the nucleic acid sequence coding for a functional    protease and a cloning site suitable for cloning the first    interaction partner into the reading frame of said functional    protease, and-   2b) a second nucleic acid construct coding for a second fusion    protein comprising the nucleic acid sequence coding for a protein    selected from the group consisting of recombinases, transcription    factors and reporter proteins,    -   a cloning site suitable for cloning the second interaction        partner into the reading frame of said protein,    -   and, where appropriate, a nucleic acid sequence coding in the        reading frame of said protein for a protein domain resulting in        the second fusion protein being anchored outside the nucleus,        and, where appropriate, which comprises expression vectors which        comprise at least one of the constructs:-   3b) a construct for expressing a recombinase gene under the control    of the functional transcription factor of No. 2;-   4b) a construct which comprises a stop cassette flanked by    recognition sites for recombinases with the downstream nucleotide    sequence of a reporter gene under the control of a constitutive    promoter.

The invention further relates to a kit for detecting and analyzingprotein interactions in a cell, which comprises expression vectorscomprising at least one of the nucleic acid constructs 1c) and 2c), ineach case under the transcriptional control of a heterologous promoter:

-   1c) a first nucleic acid construct coding for a first fusion    protein, comprising the nucleic acid sequence coding for a first    part of a protein selected from the group consisting of    transcription factors or recombinases and a cloning site suitable    for cloning the first interaction partner into the reading frame of    said protein,-   2c) a second nucleic acid construct coding for a second fusion    protein, comprising the nucleic acid sequence coding for a second    part of a protein selected from the group of transcription factors    or recombinases and a cloning site suitable for cloning the second    interaction partner into the reading frame of said protein,    -   and which comprises, where appropriate, expression vectors        comprising at least one of the following constructs:-   3c) a construct for expressing a recombinase gene under the control    of the functional, transcomplemented protein of No. 1 and 2, said    protein being a transcription factor;-   4c) a construct comprising a stop cassette flanked by recognition    sites for recombinases with the downstream nucleotide sequence of a    reporter gene under the control of a constitutive promoter.

The invention further relates to a kit for detecting and analyzingprotein interactions in a cell, which comprise at least one expressionvector comprising at least one of the nucleic acid constructs 1d) and2d) in each case under the transcriptional control of a heterologouspromoter:

-   1d) a first nucleic acid construct coding for a first fusion protein    comprising a functional enzyme from the group consisting of    proteases or recombinases and a bait protein, and-   2d) a second nucleic acid construct coding for a second fusion    protein comprising a functional inhibitor for an enzyme of the group    consisting of proteases and recombinases and a prey protein.

All of the required protein components of the methods of the inventionmay be incorporated here into the test cells both by transient and bystable transfection or infection. Incorporation into the test cells maybe carried out by way of appropriate expression vectors by any of thetransformation and transfection techniques known to the skilled workerbut, alternatively, also by infection with retroviruses or by otherviral-based methods.

The present invention furthermore also relates to the use of at leastone enzyme from the group consisting of recombinases and proteases forprotein interaction-dependent generation of a permanent and increaseddetection signal in the cell.

The embodiments and reverse embodiments described of the method of theinvention of analyzing the association and dissociation, respectively,of interaction partners or of components of a protein complex areparticularly suitable for

-   -   A) isolating unknown proteins from cDNA or ORF (open reading        frame) libraries, which interact with a known partner in a        stimulation-dependent manner, or characterizing novel        interactions of known proteins and identifying interaction        domains and regulatory mechanisms.    -   B) elucidating biological mechanisms which control the formation        of protein complexes and the dynamic composition thereof.    -   C) isolating substances which interfere directly or indirectly        with specific protein interactions, i.e. which inhibit or        promote said protein interactions.    -   D) characterizing stimulus-dependent protein-protein        interactions for a multiplicity of different signal transduction        pathways. Said interactions may then be utilized as detection        methods for activation of the corresponding cellular signaling        mechanisms or for analysis of the physiological state of cells,        for example after addition of active compounds or after a change        in culturing conditions.

The following strategies and techniques are suitable for identifying andcharacterizing novel protein interactions:

-   1.) cell lines which stably express relevant components of the    reporter system may be infected with a complex mixture of    retroviruses, said retroviruses being constructed in such a way    that, after integration, a known bait fusion protein and an unknown    prey fusion protein can be coexpressed in a cell. The prey fusion    proteins may be prepared by molecular cloning of tissue- or    celltype-specific complex cDNAs. The bait and prey fusion proteins    may be coexpressed via bicistronic RNAs, using internal ribosomal    entry sites, the IRES elements (such as, for example, the EMC    virus), (Vagner, Galy et al. 2001) (Pestova, Kolupaeva et al. 2001).    The use of bidirectional promoters likewise enables two proteins to    be simultaneously coexpressed (Baron, Freundlieb et al. 1995).    Unknown interaction partners may be identified by amplifying and    sequencing the expressed cDNAs from cells or cell clones selected by    growth or isolated by fluorescent-activated-cell-sorting (FACS).-   2.) Cell lines which stably express relevant components of the    reporter system may be infected with a complex mixture of    retroviruses, said retrovirus being constructed in such a way that,    after integration, in each case one unknown bait-prey pair is    coexpressed in a cell. The bait and prey fusion proteins may be    prepared by molecular cloning and combination of defined complex    cDNA pools. Coexpression and identification of the unknown partners    may be carried out as described under 1.).-   3.) Cell lines which stably express relevant components of the    reporter system may be infected with a complex mixture of    retroviruses, said retroviruses being constructed in such a way    that, after integration, in each case one unknown prey fusion    protein is expressed in a single cell. After transient transfection    or infection of an appropriately large number of cells with    expression plasmids coding for one or more bait fusion proteins, it    is possible to isolate novel interaction partners, as described    under 1.).-   4.) The strategy described under 3.) may also be carried out in    cells expressing no or only one component of the relevant reporter    system. In the case of transient transfection or infection of the    bait expression plasmid, the missing components must also be    introduced into the cell.-   5.) Various cell types which express, but need not necessarily    express, components of the reporter system in a stable manner may be    analyzed in high throughput transfections or transformations of    complex libraries of characterized prey expression plasmids together    with one or more known bait expression plasmids. In the case of    unmodified cells, components of the preferred reporter system may    have to be cotransfected. Complex plasmid libraries with a    continually increasing number of defined cDNAs having an open    reading frame (ORF) are offered by several suppliers (Brizuela,    Braun et al. 2001). The format of these libraries is designed in    such a way that the desired N- or C-terminal fusion proteins may be    expressed in appropriate plasmid vectors by simple recombination    (Simpson, Wellenreuther et al. 2000). Detection requires that, with    a given transfection efficiency, a sufficiently large number of    cells per reaction mixture has been transfected. Detection may be    carried out via automated image analysis, when fluorescent reporters    are used, or by established methods according to the prior art, when    enzymatic or bioluminescent reporters are used.-   6.) As described under 5.), cells may be transfected with a    combination of appropriate expression plasmids for analyzing protein    interactions, said transfection being achieved by applying the    plasmid DNA or plasmid DNA mixture to an appropriate surface and    treatment with particular transfection agents. This method is    referred to as “reverse transfection” and makes possible    simultaneous high throughput analysis of appropriate ORF expression    libraries (Ziauddin and Sabatini 2001).-   7.) It is furthermore possible, by using the detection methods    described herein, which may convert transient effects to a permanent    signal, to utilize common methods of introducing peptide or protein    fusions into cells, although the stability of exogenous proteins or    peptides in the cell may be very brief. Common methods of    introducing peptides or fusion proteins have been described by    Prochiantz (Prochiantz 2000). The appropriate fusion proteins which    can enter cells may have been modified beforehand in various ways,    for example by coupling of low molecular weight active compounds, by    coupling of particular lipids or of other substance classes.

BRIEF DESCRIPTION OF THE DRAWINGS

The method of the invention will be further illustrated by the drawings:

In order to make clear the molecular switch mechanisms, the diagrammaticdrawings depict inactive elements in gray, with activated elementshaving a black background.

In the drawings,

FIG. 1 depicts the diagrammatic representation of the plasmid constructused for the Cre recombinase-dependent two-hybrid system in mammaliancells;

FIG. 2 depicts a flowchart of the Cre recombinase-dependent two-hybridsystem for detecting constitutive protein-protein interactions;

FIG. 3 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20 after transfection with CMV-STOP/EGFP and GV;

FIG. 4 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20 after transfection with CMV-STOP/EGFP and GV, threedays after neuronal differentiation by NGF;

FIG. 5 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20.4 after transfection with GV;

FIG. 6 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20.4 after transfection with GV and BlasticidinSselection for four weeks;

FIG. 7 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20.4 after transfection with GV, G-ME2bHLH and V-ND;

FIG. 8 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20.4 after transfection with GV, G-ME2bHLH and V-ND,after addition of TSA;

FIG. 9 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20.4 after transfection with GV, G-GBR2cc, V-GBR2cc,G-GBR2ccDel and V-GBR2ccDel;

FIG. 10 depicts the evaluation of FACS analysis of the stable PC12 cellline #20.4 after transfection with GV, G-GBR2cc, V-GBR2cc, G-GBR2ccDeland V-GBR2ccDel;

FIG. 11 depicts a flowchart of the Cre recombinase-dependent two-hybridsystem for detecting induced and transient protein-protein interactions;

FIG. 12 depicts the evaluation of FACS analysis after transfection ofthe stable PC12 cell line #20.4 with a vector control, G-CREB, V-CBP/KIXand G-CREB with V-CBP/KIX under control conditions and after forskolinstimulation;

FIG. 13 depicts the diagrammatic representation of the plasmid constructused for the Cre recombinase-coupled two-switch system in mammaliancells;

FIG. 14 depicts a flowchart of the protease-dependent molecular switchfor activating a membrane-bound transcription factor;

FIG. 15 depicts the evaluaton of FACS analysis after transfection of thestable PC12 cell line #20.4 with a control, GV, TM/Tev, tevGV and TM/Tevwith tev/GV

FIG. 16 depicts the diagrammatic representation of the plasmid constructused for protein interaction-coupled transcomplementation of fragmentsof TEV protease in mammalian cells;

FIG. 17 depicts a flowchart of the protease-dependent molecular switchfor activating a membrane-bound transcription factor after proteininteraction-coupled functional reconstitution of the TEV proteasefragments;

FIG. 18 depicts the evaluation if FACS analysis after cotransfection ofthe stable PC12 cell line #20.4 with TMtevGV and with a control,TM-tev-GV, GBR1cc-CterTEV-71-243, GBR2cc-CterTEV-1-70;

FIG. 19 depicts the diagrammatic representation of the plasmidconstructs used for the protease-coupled endless switch system;

FIG. 20 depicts a flowchart of the protease-dependent molecular switchfor activating a proteolytically activatable, nonfluorescent GFPvariant, after protein interaction-coupled functional reconstitution ofthe TEV protease fragments;

FIG. 21 depicts a flowchart of the protease-dependent molecular endlessswitch for activating a proteolytically activatable, inactive TEVprotease and a proteolytically activatable nonfluorescent GFP variant,after protein interaction-coupled functional reconstitution of the TEVprotease fragments;

FIG. 22 depicts the diagrammatic representation of the plasmidconstructs used for the protease-coupled reverse switch system;

FIG. 23 depicts a flowchart of the reverse switch system after induceddissociation of the known interaction of protein X fused to a TEVinhibitor and protein Y fused to the intact TEV protease, coupled to thetwo-switch system;

FIG. 24 depicts the diagrammatic representation of the plasmidconstructs used for the protease expression feedback-coupled system forendless activation;

FIG. 25 depicts a flowchart of the protein interaction-regulatedprotease expression feedback-coupled system for endless activation.

DETAILED DESCRIPTION OF THE INVENTION

In detail, FIGS. 1 to 25 illustrate the following points andembodiments:

FIG. 1 depicts the diagrammatic representation of the plasmid constructused for the Cre recombinase-dependent two-hybrid system in mammaliancells, with, in detail,

-   -   the constructs referred to as G5 reporters, G5-bGal, G5-EGFP and        G5-Cre, expressing beta-galactosidase, the enhanced green        fluorescent protein and Cre recombinase, respectively, under the        control of a minimal promoter, the E1B-TATA box, and five        successive Gal4-dependent enhancer elements from yeast (upstream        activating sequence, UAS);    -   the constructs referred to as G5C reporters, G5C-EGFP and        G5C-Cre, expressing the enhanced green fluorescent protein and        Cre recombinase, respectively, under the control of the human        CMV minimal promoter (CMVmin) and five successive Gal4-dependent        enhancer elements from yeast (upstream activating sequence,        UAS);    -   the constructs referred to as CMV-STOP reporters, CMV-STOP/EGFP,        CMV-STOP/bGal and CMV-STOP/BlasR, being able to express the        enhanced green fluorescent protein, beta-galactosidase and the        enzyme conferring resistance to BlasticidinS under the control        of a human CMV promoter, if the STOP cassette flanked by loxP        sequence elements has been removed after Cre recombinase        activity;    -   the components of the transcription factor used for the        two-hybrid system in mammalian cells being depicted.    -   G refers to the construct for expressing the DNA-binding domain        of of the yeast Gal4 transcription factor (Gal4 DBD) under the        control of the human CMV promoter.    -   V refers to the construct for expressing the transcription        activation domain of the Herpes simplex virus protein VP16 (VP16        TAD) under the control of the human CMV promoter.    -   GV refers to the construct for expressing the fusion of the DNA        binding domain of the yeast Gal4 transcription factor (Gal4 DBD)        and the transcription activation domain of the Herpes simplex        virus protein VP16 (VP16 TAD) under the control of the human CMV        promoter.    -   GX refers to the construct for expressing the DNA-binding domain        of the yeast Gal4 transcription factor (Gal4 DBD) fused to        protein X under the control of the human CMV promoter.    -   VY refers to the construct for expressing the transcription        activation domain of Herpes simplex virus protein VP16 (VP16        TAD) fused to protein Y under the control of the human CMV        promoter.

FIG. 2 depicts a flowchart of the Cre recombinase-dependent two-hybridsystem for detecting constitutive protein-protein interactions. In thecase of a specific interaction of GX and VY which is mediated by theprotein domains X and Y, the result is functional reconstitution of atranscription factor which induces Gal4-dependent expression of the Crerecombinase. The activity of the Cre protein which is located in thenucleus results in the removal of the transcriptional inactivationelement (STOP) and in permanent activation of downstream reporter genes.

FIG. 3 depicts GFP-fluorescence and phase contrast images of the PC12cell line #20 after transfection with CMV-STOP/EGFP and transcriptionfactor GV. The cell line #20 contains the construct G5C-Cre in a stablyintegrated form and shows no EGFP expression after cotransfection withthe Cre-dependent reporter construct CMV-STOP/EGFP (bottom left). Aftercotransfection of CMV-STOP/EGFP with transcription factor GV, a GFPfluorescence can be detected (top left). The phase contrast images(right) depict a comparable number of cells.

FIG. 4 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20 after transfection with CMV-STOP/EGFP and GV, threedays after neuronal differentiation by NGF. The induction of neuronaldifferentiation and, connected therewith, prevention of further celldivisions have no influence on the activation of Crerecombinase-mediated GFP fluorescence after cotransfection ofCMV-STOP/EGFP with transcription factor GV in the PC12 cell line #20(top left). The GFP-positive cells depicted under higher magnificationshow the neuronal morphology (bottom left).

FIG. 5 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20.4 after transfection with GV. The cell line #20.4contains the constructs G5C-Cre, CMV-STOP/EGFP and CMV-TkZeo/BlasR in astably integrated form and shows GFP fluorescence only aftertransfection of transcription factor GV (top left). Under controlconditions, no GFP fluorescence is detectable (bottom left). The phasecontrast images depict a comparable number of cells (right).

FIG. 6 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20.4 after transfection with GV and BlasticidinSselection for four weeks. All cells of the BlasticidinS-resistant cellclones show a comparable GFP fluorescence, all resistant cell clones areGFP-positive.

FIG. 7 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20.4 two days after transfection with GV, G-ME2bHLH,V-ND and G-ME2bHLH with V-ND. The cell line #20.4 shows GFP fluorescenceonly after transfection of transcription factor GV (top left). Aftercotransfection of the two-hybrid interaction partners V-ND andG-ME2bHLH, no GFP-fluorescence is detectable (bottom left). Undercontrol conditions, transfection of V-ND or G-ME2bHLH, likewise no GFPfluorescence is detectable (center left). The phase contrast imagesdepict a comparable number of cells (right).

FIG. 8 depicts GFP-fluorescence and phase contrast images of the stablePC12 cell line #20.4 two days after transfection with GV, G-ME2bHLH,V-ND and G-ME2bHLH with V-ND, with addition of TSA. After transfectionof transcription factor GV, the cell line #20.4 shows GFP fluorescence(top left). After cotransfection of the two-hybrid interaction partnersV-ND and G-ME2bHLH, GFP fluorescence is likewise detectable under theTSA culturing conditions (bottom left). Under control conditions,transfection of V-ND or G-ME2bHLH, no GFP fluorescence is detectable(center left). The phase contrast images depict a comparable number ofcells (right).

FIG. 9 depicts GFP fluorescence and phase contrast images (small insetimage) of the stable PC12 cell line #20.4 after transfection with GV,G-GBR2cc, V-GBR1cc, G-GBR2cc with V-GBR1cc and G-GBR2ccDel withV-GBR1ccDel. After transfection of transcription factor GV the cell line#20.4 shows GFP fluorescence (top right). After cotransfection of thetwo-hybrid interaction partners G-GBR2cc, V-GBR1cc, GFP fluorescence islikewise detectable (bottom left). Under control conditions,transfection of an empty vector (control, top left) and of theindividually transfected interaction partners (G-GBR2cc or V-GBR1cc,bottom left), no GFP fluorescence is detectable (center left). Aftercotransfection of plasmids coding for coiled-coiled deletion mutants ofthe intracellular domains of GBR1 and GBR2, likewise no GFP fluorescenceis detectable (G-GBR2ccDel and V-GBR1ccDel, bottom inside right). Thephase contrast images depict a comparable number of cells (small insetfigure).

FIG. 10 depicts the quantitative evaluation of FACS analysis of thestable PC12 cell line #20.4 two days after transfection with GV,V-GBR1cc, G-GBR2cc and V-GBR1cc together with G-GBR2cc, depicted as therelative number of GFP-positive cells. Two days after cotransfection ofthe interaction partners G-GBR2cc and V-GBR1cc, about 100 times moreGFP-positive cells can be detected in comparison with the controls,empty vector (vector control) and the individual transfections ofG-GBR2cc and V-GBR1cc.

FIG. 11 depicts a flowchart of the Cre recombinase-dependent two-hybridsystem for detecting induced and transient protein-protein interactions.In the case of a stimulus-dependent interaction of GX and VY which ismediated by the protein domains X and Y, the result is the functionalreconstitution of a transcription factor inducing Gal4-dependentexpression of the Cre recombinase. The stimulus is represented by theexplosion symbol. The stimulation-dependent modification of proteinsection X is indicated by P. After removal of the cellular modification(represented by the X above the explosion symbol), the modification andthe protein interaction are removed. The activity of the Cre proteinwhich is located in the nucleus results in removal of thetranscriptional inactivation element (STOP) and in permanent activationof downstream reporter genes. The activity of the Cre protein may betransient and needs to reach a particular threshold only once.

FIG. 12 depicts the results of FACS analysis three days aftertransfection of the stable PC12 cell line #20.4 with a vector control,G-CREB, V-CBP/KIX and G-CREB with V-CBP/KIX, with or without forskolinstimulation. The cumulated GFP fluorescence (top) and the relativenumber of GFP-positive cells (bottom) without forskolin stimulation(framed columns) and after transient forskolin stimulation (blackcolumns) are depicted. After cotransfection of the interaction partners,G-CREB with V-CBP/KIX, the number of detected GFP-positive cells isincreased about 12 times after forskolin stimulation. The increase intotal fluorescence of the GFP-positive cells is twenty times higher thanthe unstimulated control. Transfection with V-CBP-KIX shows virtually nobackground, while transfection with the G-CREB construct has GFPbackground both in the unstimulated and in the forskolin-stimulatedreaction mixture. The likewise strong increase in GFP fluorescence afterforskolin stimulation presumably reflects the interaction withendogenously present CBP-like transcriptional cofactors.

FIG. 13 depicts the diagrammatic representation of the plasmidconstructs used for the Cre recombinase-coupled two-switch system inmammalian cells. SS refers to the signal sequence, TAG is theextracellular domain encompassing specific epitopes, TM is thePDGF-alpha receptor transmembrane domain. The constructs are describedin closer detail:

-   -   the construct referred to as renin expresses the human renin        protese under the control of the human CMV promoter.    -   the construct referred to as TEV expresses the NIa protease of        tobacco etch virus (TEV protease) under the control of the human        CMV promoter.    -   the construct referred to as TM/TEV expresses a        transmembrane-bound form of the TEV protease of the tobacco etch        virus under the control of the human CMV promoter.    -   the construct referred to as TM/S/TEV expresses a        transmembrane-bound form of the TEV protease of the tobacco etch        virus under the control of the human CMV promoter, said TEV        protease being separated from the transmembrane domain by a        peptide section (S stands for spacer).    -   the construct referred to as TM/GV expresses a        transmembrane-bound inactive form of the transcription activator        GV under the control of the human CMV promoter.    -   the construct referred to as TM/ren/GV expresses a        transmembrane-bound inactive form of the transcription activator        GV under the control of the human CMV promoter, said        transcription activator GV being separated from the        transmembrane domain by a peptide section ren. ren represents a        human renin protease-specific recognition and cleavage sequence.    -   the construct referred to as TM/tev/GV expresses a        transmembrane-bound inactive form of the transcription activator        GV under the control of the human CMV promoter, said        transcription activator GV being separated from the        transmembrane domain by a peptide section tev. tev represents a        recognition and cleavage sequence specific for the TEV protease.    -   the construct referred to as TM/Cre expresses a        transmembrane-bound inactive form of Cre recombinase under the        control of the human CMV promoter.    -   the construct referred to as TM/ren/Cre expresses a        transmembrane-bound inactive form of Cre recombinase under the        control of the human CMV promoter, said Cre recombinase being        separated from the transmembrane domain by a peptide section        ren. ren represents a recognition and cleavage sequence specific        for the human renin protease.    -   the construct referred to as TM/tev/Cre expresses a        transmembrane-bound inactive from of Cre recombinase under the        control of the human CMV promoter, said Cre recombinase being        separated from the transmembrane domain by a peptide section        tev. tev represents a recognition and cleavage sequence specific        for the TEV protease.    -   the construct referred to as TM/EYFP expresses a        transmembrane-bound form of enhanced yellow fluorescent protein        (EYFP) with C-terminal nuclear localization signals (NLS) under        the control of the human CMV promoter.    -   the construct referred to as TM/ren/EYFP expresses a        transmembrane-bound form of enhanced yellow fluorescent protein        (EYFP) with C-terminal nuclear localization signals (NLS) under        the control of the human CMV promoter, said EYFP-NLS protein        being separated from the transmembrane domain by a peptide        section ren. ren represents a recognition and cleavage sequence        specific for the human renin protease.    -   the construct referred to as TM/tev/EYFP expresses a        transmembrane-bound form of enhanced yellow fluorescent protein        (EYFP) with C-terminal nuclear localization signals (NLS) under        the control of the human CMV promoter, said EYFP-NLS protein        being separated from the transmembrane domain by a peptide        section tev. ren represents a recognition and cleavage sequence        specific for the human renin protease.

FIG. 14 depicts a flowchart of the TEV protease-dependent molecularswitch for activating a membrane-bound transcription factor andsubsequent activation of a reporter or reporter system. Coexpression ofthe TEV protease with a transmembrane-bound transcription factor (GV)which N-terminally has a TEV protease-specific recognition and cleavagesite (tev) results in the cleavage and subsequent translocalization oftranscription factor GV into the nucleus. GV-dependent reporter genes orreporter systems are activated.

FIG. 15 depicts the quantitative evaluation of FACS analysis two daysafter transfection of the stable PC12 cell line #20.4 with a controlvector (control), GV, TM/Tev, tevGV and TM/Tev together with tev/GV. Therelative number of GFP-positive cells in comparison with transfectionwith the soluble transcription factor GV is shown. The number ofdetected cells after single transfection with the empty vector, with thetransmembrane-bound TEV or with the transmembrane-bound GV is below 1%.After cotransfection of the transmembrane-bound TEV with thetransmembrane-bound transcription factor GV, the soluble GV can detecton average half of GFP-positive cells, in comparison with transfectionunder comparative conditions.

FIG. 16 depicts the diagrammatic representation of the plasmidconstructs used for protein interaction-coupled transcomplementation offragments of the TEV protease in mammalian cells, with, in detail,

-   -   the construct referred to as NterTEV expressing an N-terminal        fragment of the TEV protease of tobacco etch virus under the        control of the human CMV promoter,    -   the construct referred to as CterTEV expressing an N-terminal        fragment of the TEV protease of tobacco etch virus under the        control of the human CMV promoter,    -   the construct referred to as NterTEV-X expressing an N-terminal        fragment of the TEV protease of tobacco etch virus as fusion        protein with the N terminus of a protein X under the control of        the human CMV promoter,    -   the construct referred to as CterTEV-Y expressing a C-terminal        fragment of the TEV protease of tobacco etch virus as fusion        protein with the N terminus of a protein Y under the control of        the human CMV promoter,    -   the construct referred to as X-NterTEV expressing an N-terminal        fragment of the TEV protease of the tobacco etch virus as fusion        protein with the C terminus of a protein X under the control of        the human CMV promoter,    -   the construct referred to as Y-CterTEV expressing a C-terminal        fragment of the TEV protease of the tobacco etch virus as fusion        protein with the C terminus of a protein Y under the control of        the human CMV promoter.

FIG. 17 depicts a flowchart of the protease-dependent molecular switchfor activating a membrane-bound transcription factor after proteininteraction-coupled functional reconstitution of TEV protease fragments.After coexpression of plasmids which, on the one hand, code for anN-terminal TEV fragment-protein X fusion protein and for a C-terminalTEV fragment-protein Y fusion protein, the proteolytic activity isfunctionally reconstituted in the case of the specific interaction ofthe proteins or protein sections X and Y. This is followed by a stablyexpressed or transiently cotransfected membrane-bound transcriptionfactor (GV) which contains a TEV-specific recognition and cleavagesequence (tev) being detached from the membrane. The result istranslocalization of GV into the nucleus and activation of a reportergene or reporter system.

FIG. 18 Part A depicts the quantitative evaluation of FACS analysis twodays after transfection of the stable PC12 cell line #20.4 with anexpression vector coding for the membrane-bound transcription factor GVcontaining an N-terminal TEV-specific recognition and cleavage sequence(TM-tev-GV). The cumulated fluorescence relative to the activity of themembrane-bound TEV protease is shown in %. Expression vectors werecotransfected with TMtevGV, which code for:

-   -   control: membrane-bound TEV (TM-TEV),    -   a membrane-bound GBR1cc domain fused to a C-terminal TEV        protease fragment, from amino acid 71-243        (GBR1cc-CterTEV-71-243),    -   a membrane-bound GBR2cc domain fused to an N-terminal TEV        protease fragment, from amino acid 1-70 (GBR2cc-CterTEV-1-70)        and    -   a membrane-bound GBR1cc domain fused to a C-terminal TEV        protease fragment, from amino acid 71-243        (GBR1cc-CterTEV-71-243) together with a membrane-bound GBR2cc        domain fused to an N-terminal fragment of said TEV protease,        from amino acid 1-70 (GBR2cc-CterTEV-1-70).

After cotransfection of the TEV fragments as fusion proteins of theinteraction domains GBR2cc and GBR1cc about 30% of the activity of themembrane-bound TEV protease is obtained.

B depicts combinations of further TEV fragments fused to the coiled-coilinteraction domains of GBR1 and GBR2. The constructs were cotransfectedinto PC12 cellls with the membrane-anchored TM-tev-GV, a plasmidtranscriptionally activatable by GV and coding for Cre recombinase, anda reporter plasmid coding for firefly luciferase under the control of astrong promoter. However, transcription of said luciferase wasinterrupted by a stop cassette located between gene and promoter andflanked by pLox. The system was activated by specifictranscomplementation of the TEV protease, ultimately leading toproduction of luciferase at the end of the cascade. The bar diagramdepicts this activity in RLU (relative luminescence units) and comparesit with the signal of the intact protease.

FIG. 19 depicts the diagrammatic representation of the plasmidconstructs used for the protease-coupled endless switch system, with, indetail,

-   -   the construct referred to as TtevEV being an inactive TEV        protease under the control of the human CMV promoter. Insertion        of a peptide sequence containing at least one TEV-specific        recognition and cleavage sequence renders this modified TEV        protease inactive. After specific proteolytic cleavage by an        active TEV protease, the modified TEV protease TtevEV is        activated,    -   the construct referred to as EYtevFP being an inactive,        nonfluorescent enhanced green fluorescent protein (EYFP) under        the control of the human CMV promoter. Insertion of a peptide        sequence containing at least one TEV specific recognition and        cleavage sequence renders this modified EYFP inactive. The        modified EYtevFP is activated after specific proteolytic        cleavage by an active TEV protease.

FIG. 20 depicts a flowchart of the protease-dependent molecular switchfor activating a proteolytically activatable nonfluorescent EYFP variantafter protein interaction-coupled functional reconstitution of TEVprotease fragments. After coexpression of plasmids which, on the onehand, code for an N-terminal TEV fragment-protein X fusion protein andfor a C-terminal TEV fragment-protein Y fusion protein, the proteolyticactivity is functionally reconstituted in the case of specificinteraction of the proteins or protein sections X and Y. A stablyexpressed or transiently cotransfected inactive, proteolyticallyactivatable reporter protein, such as, for example, an appropriatelymodified EYFP (EY-tev-FP), is then specifically proteolytically cleavedand activated.

FIG. 21 depicts a flowchart of the protease-dependent molecular endlessswitch for activating a proteolytically activatable, inactive TEVprotease and a proteolytically activatable, nonfluorescent GFP variantafter protein interaction-coupled functional reconstitution of TEVprotease fragments. After coexpression of plasmids which, on the onehand, code for an N-terminal TEV fragment-protein X fusion protein andfor a C-terminal TEV fragment-protein Y fusion protein, the proteolyticactivity is functionally reconstituted in the case of specificinteraction of the proteins or protein sections X and Y. A stablyexpressed or transiently cotransfected inactive, proteolyticallyactivatable TEV protease (TtevEV) is then specifically proteolyticallycleaved and permanently activated. Subsequently, a stably expressed ortransiently cotransfected inactive, proteolytically activatable reporterprotein such as, for example, an appropriately modified EYFP (EY-tev-FP)is specifically proteolytically cleaved and activated. Activation of theconstitutively expressed proteolytically activatable functional elementsresults in a molecular endless loop.

FIG. 22 depicts the diagrammatic representation of the plasmidconstructs used for the protease-coupled reverse switch system, with, indetail,

-   -   the construct referred to as TEVInh expressing a protein        inhibitor or peptide inhibitor of the TEV protease of tobacco        etch virus under the control of the human CMV promoter.    -   the construct referred to as TEV-X expressing a fusion protein        of the intact TEV protease of tobacco etch virus and a protein X        under the control of the human CMV promoter. The protein X is        fused to the C terminus of the TEV protease.    -   the construct referred to as TEVInh-Y expressing a fusion        protein of a TEV inhibitor and a protein Y under the control of        the human CMV promoter.

The protein Y is fused to the C terminus of the TEV inhibitor.

-   -   the construct referred to as X-TEV expressing a fusion protein        of the intact TEV protease of tobacco etch virus and a protein X        under the control of the human CMV promoter. The protein X is        fused to the N terminus of the TEV protease.    -   the construct referred to as Y-TEVInh expressing a fusion        protein of a TEV inhibitor and a protein Y under the control of        the human CMV promoter. The protein Y is fused to the N terminus        of the TEV inhibitor.

FIG. 23 depicts a flowchart of the reverse switch system after induceddissociation of the known interaction of protein X fused to a TEVinhibitor and of protein Y fused to the intact TEV protease, coupled tothe two-switch system. The protein X and protein Y-mediated interactionof the TEV inhibitor with the intact TEV protease results ininactivation of the TEV protease. After induced removal of theinteraction, represented by the explosion symbol, the TEV protease isactivated. The downstream reporter system depicted here is thetwo-switch system (see FIG. 14).

FIG. 24 depicts the diagrammatic representation of the plasmidconstructs used for the protease expression feedback-coupled system forendless activation, with, in detail,

-   -   the constructs referred to as G5-TEV expressing the TEV protease        under the control of a minimal promoter, the E1B-TATA box, and        five successive Gal4-dependent enhancer elements from yeast        (upstream activating sequence, UAS).    -   the constructs referred to as G5C-TEV expressing the TEV        protease under the control of the human CMV minimal promoter,        and five successive Gal4-dependent enhancer elements from yeast        (upstream activating sequence, UAS).

FIG. 25 depicts a flowchart of the protein interaction-regulatedprotease expression feedback-coupled system for endless activation.After coexpression of plasmids which, on the one hand, code for anN-terminal TEV fragment-protein X fusion protein and for a C-terminalTEV fragment-protein Y fusion protein, the proteolytic activity isfunctionally reconstituted in the case of specific interaction of theproteins or protein sections X and Y. A stably expressed or transientlycotransfected membrane-bound transcription factor (GV) containing aTEV-specific recognition and cleavage sequence (tev) is then detachedfrom the membrane. The result is a translocalization of GV into thenucleus and activation of two coregulated or independent reporter genesone of which is the intact TEV protease. The GV-regulated expressed TEVprotease results in further cleavage of the constitutively expressedTM-tev-GVs and results in permanent activation of the complete reportersystem.

The following examples describe the individual embodiments of the methodof the invention in more detail.

All molecular cloning and transfections were carried out using standardprotocols according to Sambrook et al (Sambrook-J and Russell-D W 2001).

EXAMPLE 1 Preparation of the Plasmid Vectors of the CreRecombinase-Based Reporter System

The functional elements of the plasmid vectors for carrying out the Crerecombinase-based reporter system and the application in the two-hybridsystem in mammalian cells are diagrammatically depicted in FIG. 1.

Construction of the reporter plasmids: the plasmid G5-CAT carries, 5′ ofthe chloramphenicol transferase (CAT) reporter gene, the TATAA box ofthe human E1B gene and five successive enhancer elements (upsteamactivating sequence, UAS) having the optimal recognition sequence forthe Saccharomyces cerevisiae Gal4 transcription factor. The G5-CATplasmid DNA served as starting vector for preparing the G5 reportersused and was cut using combinations of restriction enzymes in such a waythat it was possible to remove the CAT gene 5′ and 3′ from the vectorbackbone and to insert the appropriately prepared reporter gene DNAfragments. For this purpose, the Cre recombinase of bacteriophage P1(Cre) was amplified by the polymerase chain reaction (PCR) usingspecific oligonucleotides and modified 5 by a start codon flanked by theKozak sequence (Kozak 1989) (Kozak 1987). The plasmid vectors G5C-EGFPand G5C-Cre were cloned using the plasmids tetO-EGFP and tetO-Cre andG5-Cat as backbone. The E1B-TATAA box and the CAT reporter were removedand replaced with the human cytomegalievirus (CMV) minimal promotor andthe corresponding reporter gene.

Coding regions of the DNA binding domain (DBD) of the yeasttranscription factor Gal4 and of the transactivation domain (TAD) of theHerpes simplex protein VP16 were amplified by means of PCR fromcorresponding yeast two-hybrid vectors and cloned into the eukaryoticexpression vectors pCMV. The oligonucleotides were designed so as tointroduce 5′ a restriction cleavage site and a Kozak sequence-flankedATG and for the last codon 3′ to be in the reading frame with anotherintroduced restriction cleavage site without stop codon. Stop codons ofthe three possible reading frames are located in the vector pCMV 3′ ofthe multiple cloning sequences (MCS) so that it was possible to utilizethe vectors pCMV-Gal4 DBD (G) and pCMV-VP16 TAD (V) (see FIG. 1) asstarting vectors for C-terminal fusions with further proteins or proteinsections. The vector GV which codes for a fusion protein of Gal4 DBD andVP16 TAD was prepared starting from the pCMV-Gal4 plasmid vector and thePCR product coding for V16 TAD, taking into account a continuous readingframe (see FIG. 1).

The Cre-activatable CMV-STOP/REPORTER constructs were prepared bysequential cloning into pCMV. First, the STOP cassettes were generatedby PCR, incorporating in each case two loxp sites (recognition andrecombination elements of Cre recombinase) in the same orientation 5′and 3′ of a neomycin resistance- and of a zeocin-resistance-conferringelement (neoR and TKZeoR, see FIG. 1). The corresponding reporter genedownstream of. Cre was then cloned in 3′ of the STOP cassette. EGFP andbGal were introduced 3′ of the neoR STOP cassette and an elementconferring resistance to BlasticidinS was introduced 3′ of the TKZeoRSTOP cassette. The functionality of the STOP-REPORTER cassettes wasanalyzed via transient transfections in Cos7 cells. For this purpose,said cassettes were cotransfected together with a control vector or witha CMV-Cre plasmid vector, it being possible to observe the activity ofthe downstream reporters, EGFP, bGal and BlasR, only aftercotransfection with CMV-Cre.

EXAMPLE 2 Functional Analysis of the Components of the CreRecombinase-Based Reporter System Via Transient Transfection into PC12and Cos7 Cells

The reporter plasmids or combinations of reporter plasmids were testedvia transient transfections into the PC12 and Cos7 cell lines. For thispurpose, between 10⁵ and 10⁶ PC12 or Cos7 cells were electroporatedusing a GenePulser II with Capacity Extender module (BioRad, Munich,Germany). Plasmid DNA was purified using the Qiafilter method (Qiagen,Hilden, Germany). For each electroporation, a total of 5 μg of plasmidDNA was always used, filling up with plasmid DNA of an empty vector,depending on the reaction mixture. The electroporation was carried outin special cuvettes (PeqLab) in the appropriate cell growth medium andusing the following parameters: Cos7 cells, 10⁵ cells in 300 μl perreaction mixture, pulses with 250 mV at 500 μF; PC12 cells, 10⁶ cells in300 μl per reaction mixture, pulses of 220 mV at 960 μF. Afterelectroporation, the cells were transferred to 6 cm or 24 well cellculture dishes and cultured. Analysis was carried out usually 12-72 hafter transfection, depending on the reporter used, using fluorescencemicroscopy, FAC sorting (EGFP) or by colorimetric detection with X-Galin fixed cells (bGal). The average transfection efficiency was about 40%for Cos7 cells and about 30% for PC12 cells. The amount of DNA of thereporter plasmids G5-bGal, G5-EGFP, G5C-EGFP, CMV-STOP/EGFP andCMV-STOP/bGal was always 1 μg per reaction mixture, with the amounts ofthe Cre recombinase reporters, G5-Cre and G5C-Cre being varied. Thefunctionality of the reporters was tested via cotransfection with theexpression plasmid of the complete transcription activator GV (1 μg perreaction mixture). The results were comparable between PC12 and Cos7cells, with a slightly stronger background but overall higher signalintensity in Cos7 cells compared to PC12 cells.

The Cre-based reporter system used herein is based on the Gal4-dependenttranscriptional activation of a Cre reporter plasmid. The expressed Creprotein can then catalyze the excision of a transcriptional STOPcassette flanked by Cre recognition and recombination sequences (loxpsites) in the same orientation. The activated reporter gene is now underthe control of the constitutive human CMV promoter which is very strongin most cell lines, resulting in an enormous increase in the signal.

After cotransfection of GV with the G5-bGal reporter, X-Gal staining wasdetected in a multiplicity of cells after only 12 h. Aftercotransfection of GV with the G5-EGFP reporter, GFP fluorescence wasdetected only in Cos7 cells in a small number of cells after 72 h.Transfections of the G5-bGal and G5-EGFP reporters exhibited nobackground activity. After cotransfection of the G5C-EGFP reporterplasmid with GV, a markedly higher number of GFP-positive cells,compared to the control transfection without GV, was detected after only48 h. Some GFP-positive cells, however, were also detected in thecontrol transfection. Transfection of GV together with CMV-STOP/EGFP orCMV-STOP/bGal exhibited no background. Cotransfection of in each case 1μg of G5-Cre and CMV-STOP/EGFP showed a very high number of GFP-positivecells after 48 h. The first GFP signals were detected after only 12 h.The best ratio of GV-induced signal to background was obtained bytransfection of 50 ng of G5-Cre with in each case 1 μg of CMV-STOP/EGFPor CMV-STOP/bGal and GV. The increased basal promoter activity of theG5C-Cre construct made it impossible to reduce the background byreducing the amounts used, as for G5-Cre.

The evaluation of the transient transfections for analyzing thecomponents of the Cre-based reporter system showed the following:

-   1). The reporter system is characterized by a very high    sensitivity. 2) Due to said high sensitivity, it is not possible to    completely remove the background of components of the system in    transient transfections. 3) Using the Cre recombinase, it is    possible to use EGFP as downstream Cre reporter with a sensitivity    and kinetics comparable to bGal. 4) When using a relatively strong    basal minimal promoter (CMVmin), EGFP is also less sensitive as    reporter than bGal.

EXAMPLE 3 Application of the Cre-Recombinase-Based Reporter System AfterTransient Transfection into PC12 and Cos7 Cells as two-Hybrid System forAnalyzing Constitutive Protein Interactions

The application of the Cre recombinase-based reporter system in thetwo-hybrid system in mammalian cells was tested via analysis of knowninteraction partners (see FIG. 2, flowchart of the Crerecombinase-dependent two-hybrid system). Most basic helix-loop-helix(bHLH) proteins form heterodimeric complexes. The interaction ismediated via two amphipathic helices which form a characteristicfour-helix bundle (Ma, Rould et al. 1994) (Baxevanis and Vinson 1993).The interaction between the bHLH proteins ME2 and Nex and, respectively,NeuroD served as a test system. For this purpose, the bHLH domain of ME2was amplified by PCR and expressed as fusion protein with Gal4-DBD(G-ME2bHLH). Full length NEX and NeuroD were expressed as fusionproteins with VP16 TAD (V-ND and V-Nex, respectively).

Another known motif which mediates specific protein-protein interactionsis the leucine zipper motif in coiled-coil (cc) domains (Lupas 1996).Another test system used were parts of the intracellular sections ofGBR1 and GBR2 which in each case contain a cc domain via which they formheterodimers (Kuner, Kohr et al. 1999). GBR1cc (L859-K960) and GBR2cc(1744-G849) were amplified and cloned by means of PCR using specificoligonucleotides. GBR1cc was fused to the C terminus of VP16 (V-GBRlcc),and GBR2cc was fused to the C terminus of Gal4 (G-GBR2cc). Deletionmutants of said protein domains, GBR1 (L859-K960 AS887-L921) and GBR2(1744-G849AS785-Q816) (V-GBR1ccDel and G-GBR2ccDel) were used asnegative control, and these mutations had previously been shown, byimmunoprecipitation and by means of yeast two-hybrid technique, not tointeract.

An interaction was detected both for the interaction partners G-ME2bHLHand V-NEX and, respectively, V-ND and for G-GBR2cc and V-GBR1cc in PC12and Cos7 cells, using the Cre reporter system and GFP fluorescence asmeasure. The controls, individual transfections and the coiled-coildeletion constructs (V-GBR1ccDel and G-GBR2ccDel), showed no orsubstantially weaker signals. The following relative strength ofinteractions was obtained from the experiments:GBR2ccV-GBR1cc>>G-ME2bHLH/V-ND>>G-ME2bHLH/V-NEX. The results wereconfirmed using bGal as two-hybrid reporter.

EXAMPLE 4 Preparation of stable PC12 and Cos7 Cell Lines Containing theComponents of the Cre Reporter System

The results from the experiments of the Cre recombinase-based two-hybridsystem after transient transfection in mammalian cells indicated that 1)the sensitivity of the system was comparable to a beta-galactosidasereporter, even when using an EGFP downstream of Cre, and 2) owing to theincreased sensitivity and to the switch mechanism of the Cre activity,it was not possible to completely reduce the background. In order to beable to control the background of the system, first a component of thetetracyclin-dependent gene regulation system, which enables expressionof the interaction partners to be finally regulated, was stablyincorporated into PC12 and Cos7 cells (Gossen, Freundlieb et al. 1995).For this purpose, a DNA fragment coding for the tet-dependenttransactivator (tTA) under the control of the CMV promoter wascotransfected with a linearized DNA element, neoR, which confersresistance to the aminoglycoside G418. G418 selection (400 μg/ml) wasstarted three days after transfection, and after 3-4 weeks cell cloneswere identified and isolated. The latter were analyzed independently forfunctional tTA expression via cotransfection with a tTA-dependentreporter (tetO-EGFP). In each case one PC12 cell clone and one Cos7 cellclone with tetracycline-dependent regulation of the GFP reporter wereused for the further steps.

In the next step, the G5-Cre or G5C-Cre DNA fragments were cotransfectedwith a fragment conferring resistance to HygromycinB. Four weeks afterHygromycinrB selection (60 μg/ml), cell clones were isolated andanalyzed for GV-dependent Cre activity. For this purpose, the Cos7 andPC12 cell clones were in each case transfected with CMV-STOP/EGFP andcotransfected with CMV-STOP/EGFP and GV and analyzed forGV/Cre-dependent induction of GFP fluorescence. All of the Cos7 and mostof the PC12 cell clones which showed an increase in Cre activity due toGV likewise exhibited a certain Cre background activity. PC12 cell clone#20 showed absolutely no constitutive Cre expression, i.e. noGFP-positive cell was detectable after transfection with CMV-STOP/EGFP(see FIG. 3, bottom left). After cotransfection of CMV-STOP/EGFP and GVon the other hand, a multiplicity of GFP-positive cells were detected(see FIG. 3, top left).

PC12 cells are an established cultured cell line of a ratpheochromocytoma and thus originate from sympathoadrenergic tissue ofthe adrenal medulla (Greene and Tischler 1976). These cells can bestimulated with nerve growth factor (NGF) and differentiate in anNGF-dependent manner to a neuron-related cell type which is postmitoticand forms neuronal processes. In order to test the Cre-based reportersystem under these postmitotic conditions, GV-transfected PC12 cells ofthe line #20 were stimulated with NGF (2.5 S NGF, 5 ng/ml, Promega)immediately after transfection and analyzed three days after GV-inducedGFP-fluorescence (see FIG. 4). The efficiency of the Cre reporter system(lower magnification, FIG. 4 top) was not influenced by NGF-induceddifferentiation (lower magnification, FIG. 4 top). The neuronalmorphology of the cells was not impaired by the Cre reporter system(lower magnification, FIG. 4 top). In summary, these results demonstratethat the Cre reporter system functions in stable PC12 cell clones in abackground-free manner, even under postmitotic conditions.

In order to stably express all necessary components of the Cre-basedreporter system, PC12 cells of the line #20 were cotransfected with thelinearized plasmid constructs CMV-STOP/EGFP (STOP=neoR) andCMV-STOP/BlasR (STOP=TKZeoR) and selected with zeocin (500 μg/ml,Invitrogen). GFP-negative and blasticidinS-sensitive cell clones wereanalyzed via transfection with GV. GFP-positive cells of the cell clone#20.4 were detected two days after GV transfection (see FIG. 5). Threeto four weeks after GV transfcktion and blasticidinS selection (2 μg/ml,Calbiochem), resistant cell clones were identified, and all cells ofthese clones were GFP-positive (see FIG. 6). In summary, theseexperiments demonstrate that the cell clone 20.4 has stably integratedand functionally expressed all components of the Cre-based reportersystem.

EXAMPLE 5 Application of the Two-Hybrid System in the PC12 Cell Line20.4 for Analyzing Constitutive Protein Interactions

As described in example 4, the PC12 cell line 20.4 expresses allcomponents of the Cre-based reporter system in a completely functionalmanner. In order to test whether the cell line 20.4 can be utilized forapplication in the two-hybrid system (see FIG. 2), the known interactionpartners described in example 3 were analyzed. Two days aftercotransfection of the interaction partners G-ME2bHLH and V-ND, noGFP-positive cells were detectable (see FIG. 7, bottom left). Aftersingle transfections of G-ME2bHLH and V-ND, GFP-positive cells werelikewise not detectable (see FIG. 7, central figures, left). Thecontrol, after transfection with GV, showed the expected high number ofGFP-positive cells (see FIG. 7, top left). This result, together withthe observation of complete absence of constitutive background withrespect to uninduced Cre expression, suggested that at least theGal4-dependent Cre reporter had been integrated into a region of theheterochromatin and was thus accessible only by very strongtransactivators such as GV. Therefore, the same two-hybrid analysis wascarried out in the following experiments, after transient addition oftrichostatin A (3 μM, for 12 h, Sigma) (FIG. 8). TSA acts as aninhibitor of deacetylases, and inhibition of deacetylation results in anoverall less densely packed, transcriptionally inactive heterochromatinsections. After transient addition of TSA into the culturing medium ofPC12 20.4 cells, a multiplicity of GFP-positive cells were now detected,two days after transfection of the interaction partners G-ME2bHLH andV-ND (see FIG. 8, bottom left). After single transfections of G-ME2bHLHand V-ND, no GFP-positive cells were detectable (see FIG. 8, centralfigures, left). The control, after transfection with GV, showed theexpected high number of GFP-positive cells (see FIG. 8, top left). Therelatively high concentration of 3 μM TSA for a period of 12 h, however,also resulted in a lower number of surviving cells (cf. FIG. 7 and FIG.8, right-hand column). The TSA concentration with the best ratio of cellsurvival and nearly background-free detection of the interaction ofG-ME2bHLH and V-ND, was 300 μM for 12 h. For the slightly strongerinteraction of GBR2cc and V-GBRlcc (see example 3), the experiments werecarried out with TSA concentrations of 200 μM TSA for 12 h (see FIG. 9).Only after cotransfection of the interaction partners GBR2cc andV-GBR1cc, a multiplicity of GFP-positive cells were detected, aftersingle transfection and cotransfection of the cc deletion mutants,GBR2ccDel and V-GBR1ccDel, no GFP-positive cells were microscopicallydetectable (see FIG. 9, bottom bar). Analysis by Fluorescent ActivatedCell Sorting (FACS) confirmed these results (see FIG. 10).

In summary, these results demonstrate that it is possible to carry out atwo-hybrid analysis in the PC12 cells of the line 20.4 and to controlthe sensitivity and, respectively, the background by addition of TSA.

EXAMPLE 6 Application of the Cre Recombinase-Based Reporter System asTwo-Hybrid System for Analyzing Induced and Transient Interactions

This example describes utilization of the Cre recombinase-based reportersystem in the two-hybrid approach of detecting a stimulus-induced andtransient interaction in vivo. As FIG. 11 diagrammatically shows,transient activation of the Cre reporter is sufficient in order toensure, via the function of the Cre recombinase located in the nucleus,permanent activation of downstream reporter.

A well-characterized example of an induced protein-protein interactionis the phosphorylation-dependent binding of the transcription activatorsCREB to the transcription coactivator CBP (CREB Binding Protein)(Chrivia, Kwok et al. 1993). For example, protein kinase A(PKA)-mediated phosphorylation of CREB at Ser133, in the“kinase-inducible-domain (KID)”, results in specific binding to the“KIX” domain of CBP. PKA may be stimulated by adding the adenylatecyclase-stimulating substance forskolin, leading to a transient increasein the intracellular cAMP level and thus to PKA activation. Afterremoving PKA stimulation by removing the forskolin from the culturemedium of cells, CREB-Serl33 is rapidly dephosphorylated via activephosphatases endogenous to the cell. For analysis in the two-hybridsystem in the PC12 20.4 cells, CREB was fused C-terminally to Gal4 DBD(G-CREB), and the CBP-KIX domain was fused to the C terminus of VP16 TAD(V-CBP-KIX).

Three days after transfection of PC12 20.4 cells with G-CREB andV-CBP-KIX and corresponding controls, detection of the interaction wasanalyzed by FACS. Without forskolin stimulation, neither single norcotransfection of the constructs resulted in significant activation ofthe Cre/EGFP reporter system (see FIG. 12, top and bottom graph, framedbars). After transient forskolin stimulation immediately aftertransfection (4 μM for 12 h) and FACS analysis after 3 days, a distinctincrease in the total number of GFP-positive cells and in the cumulatedtotal fluorescence after cotransfection of G-CREB and V-CBP-KIX wereobserved (see FIG. 12, black bar, right). A by far smaller but likewisesignificant increase in the number of GFP-positive cells and totalfluorescence was measured in a forskolin-dependent manner for singletransfection with G-CREB. This can be explained by the likewisestimulated interaction of G-CREB with the endogenous CBP or relatedtranscriptional cofactors. The stronger relative increase in totalfluorescence with and without stimulation, in comparison with thesomewhat smaller relative increase in GFP-positive cells indicates akinetic component in the system.

In summary, these results demonstrate that it is also possible to carryout two-hybrid analyses in the PC12 20.4 cells, with interactions whichare stimulus-dependent and transient. How transient said interaction is,was shown by Chawla and Bading. Their analyses of CREB phosphorylationafter short-time calcium signals revealed that S133 phosphorylationresults in rapid activation of CREB, but the protein is inactive againdue to dephosphorylation only after a few minutes (Chawla and Bading,2001) (dephosphorylation of S133 results in dissociation of CREB andCBP-KIX).

EXAMPLE 7 Proteolytic Activation of a Membrane-Anchored TranscriptionActivator After Transient Cotransfection and, Connected Therewith,Activation of the Reporter System in PC12 20.4.

Membrane anchoring of the Gal4/VP16 fusion protein in PC12 20.4 andactivation of the reporter system after proteolytic removal.

In this example, the feasibility in principle of the protease switch onthe membrane in PC12 20.4 cells is described. It was the aim toestablish a further intracellular mechanism which transduces proteolyticevents at the periphery into permanent signals. For this purpose, theGal4/VP16 fusion protein required for recombinase activation wasanchored on the membrane (TM-GV) and was then intended to activate via aspecific proteolytic cleavage the reporter system in the nucleus. Stablelocalization of Gal4/VP16 on the cell membrane was achieved by fusion tothe transmembrane domain of the PDGF (platelet derived growth factor)receptor, with insertion and correct orientation of the construct in themembrane being ensured by an N-terminal signal sequence. The efficacy ofactivation was analyzed via expression of the EGFP reporter. For thispurpose, the transfected cells were trypsinated after 48 h and theproportion of positive cells was quantitatively determined in an FACanalyzer (FACSCalibur from BD Bioscience). For this experiment, in eachcase 1.5×10⁵ PC12 20.4 cells were plated on a 24-well plate andtransfected with in each case 0.5 μg of the corresponding plasmid DNA onthe next day (Lipofectamine2000; Invitrogen). Transient expression ofthe chimeric membrane protein in PC12 20.4 cells resulted in nosignificant activation of the reporter system (FIG. 15), demonstratingthat the GV transcription activator is stably anchored on the membrane.To proteolytically remove the GV transactivator, the bases coding forthe 7 amino acid recognition sequence (ENLYFQG) (SEQ ID NO: 1) of thetobacco etch virus (TEV) Nla protease (TEV protease) were inserted intothe DNA sequence between PDGF transmembrane segment and Gal4/VP16.Introduction of this or alternative protease cleavage sites did notresult in any unspecific release of the Gal4/VP16 fusion protein.Coexpression of the TEV protease, however, led to efficient cleavage ofthe TM/tev/GV construct and subsequently to distinct activation of theCre/EGFP reporter system. In another step, it was intended todemonstrate that the TEV protease is active even after membraneanchoring. The latter is a basic requirement for the interactionanalysis of membrane proteins. For this purpose, the TEV protease was,analogously to the Gal4/VP16 reporter, N-terminally fused to thetransmembrane domain of the PDGF receptor and coexpressed with theTM/tevS/GV construct in PC12 20.4 cells. The result showed nosignificant difference in the activation of the Cre/EGFP reporter systemby soluble and membrane-bound protease. This example underlines thesuitability in principle of the method of detecting protein interactionsoutside the nucleus, in particular on the cell membrane. A preconditionfor this is the functional coupling of an interaction to the proteolyticcleavage, and this may be carried out by transcomplementation of aprotease or, in the case of a low concentration of the partnersinvolved, also by producing a suitable proximity between protease andcleavage site (FIG. 15).

Analogously to the Gal4/VP16 transcription activator construct, the Crereporter was anchored directly on the cell membrane, with theproteolytic release thereof then leading directly to EGFP activation inthe nucleus. In the case of weak interactions of very rare proteins, itis possible that the double strategy described is not sensitive enoughin order to transduce an interaction on the membrane into a signal. Asubstantially higher sensitivity is achieved, if, instead of theGal4/VP16 activator used before, the Cre recombinase is coupled directlyto the membrane by means of a PDGF transmembrane domain and linked by aprotease recognition site. Overexpression of the TM-Cre constructinitially resulted in increased background activity and had to becompensated for by weaker expression. For this purpose, the TM-Creconstruct was stably transfected into PC12 cells and selected forbackground-free clones. These cell lines, cotransfected with TEVprotease and STOP-EGFP reporter plasmid, subsequently turned rapidly anddistinctly green.

EXAMPLE 8 Transcomplementation of TEV Protease

Transcomplementation of the TEV protease provides a possibility ofconverting a protein interaction into the proteolytic removal of amembrane-bound transcription activator. The Nla protease of tobacco etchvirus is a member of the family of C4 cysteine peptidases which arestructurally homologous to the trypsin-like serine proteases. They havetherefore likewise a bilobal β-barrel structure in which three aminoacids are characteristically arranged. Modeling of the TEV proteasesequence to a known 3D structure of a related protease (Dengue virus NS3protease PDB 1bef) with the aid of the Swissmodel Software suggests thatthese amino acids, the “triads”, are spread over the two lobes of thestructure and are located opposite each other. Although the two lobesare physically connected with one another, they seem to foldindependently of one another, however. The starting point fortranscomplementation was the aim to separate the amino acids of thetriads in such a way that they are located on different fragments whichcould per se form a tertiary structure but which exhibit no activity.The DNA for the N- and C-terminal fragments of the TEV protease wasamplified by means of specific PCR oligonucleotides, introducing 5′ anNheI and a 3′ KpnI restriction cleavage site, and cloned in a plasmidvia NheI and KpnI to the 3′ termini of the “coiled coil” domains of GBR1and GBR2, which are anchored via the PDGF transmembrane domain (seeexamples 3 and 5). These constructs were designed in such a way that theinteraction of the membrane-anchored “coiled-coil” regions recombine theN- and C-terminal fragments of the TEV protease to an active form, andthis may be detected via removal of the likewise membrane-anchoredGal4/VP16 transactivator in PC12 20.4 cells. In order to ensure thatnone of the two generated TEV “subunits” has proteolytic activity, theywere independently transfected and tested. It was also checked, bycloning the particular GBR deletion mutants (see example 5), that the C-and N-terminal lobes do not gain activity after cotransfection. Theexperiment revealed that several regions in the TEV protease aresuitable for transcomplementation, in particular the region betweenamino acid 60 and 80 and, in particular, the region between 95 and 120.Dividing the protease at these sites led to two inactive fragments, andcoexpression of these variants fused to interaction domains in manycases reconstituted the proteolytic activity, with some examples beingdescribed in more detail below. The two TEV fragments Gly1-Thr70 andThr71-Gly243 gained more than 30% of the activity of the intact proteasewhen fused to the membrane-anchored, interacting “coiled coil” domainsof GBR1 and GBR2 (FIG. 18). Neither individual expression norcoexpression of the fragments fused to the noninteracting mutants of theGBR domains, resulted in comparably strong activation of the reportersystem (FIG. 18 a). A combination of the partially overlapping fragmentsGly1-Thr70 and His61-Gly243 exhibited a similarly good activation. Partsof a protein intended to result in a functional complete protein bytranscomplementation may therefore also partly overlap.

Several fragments which gain activity after interaction were found inthe region of position 100 (the predicted linker domain of N-terminaland C-terminal lobe). The combination of fragments Gly1-T118 andK119-Gly243, in particular, distinguished itself by very high activityafter transcomplementation (FIG. 18 b).

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1. A method of detecting and analyzing protein interactions in a cell,which comprises the steps: a) expressing in the cell a1) a first fusionprotein comprising a first interaction partner and a part of the 27 kDaNIa protease of tobacco etch virus, wherein the part of the NIa proteasealone does not have proteolytic activity, and a2) a second fusionprotein comprising a second interaction partner and another part of saidNIa protease, wherein said another part of the NIa protease alone doesnot have proteolytic activity, and a3) a reporter protein, whosereporter activity can be activated or inactivated by proteolysis, b)reconstituting the functional NIa protease activity via interaction ofsaid first and second interaction partners, c) detecting and analyzingactivation of the proteolysis-activatable or inactivation of theproteolysis-inactivatable reporter protein by the reconstitutedfunctional NIa protease of step b), wherein said reporter protein isselected from the group consisting of recombinases, transcriptionactivators, fluorescent proteins, luciferases, beta-galactosidase,alkaline phosphatases, beta-lactamase, proteins and enzymes conferringresistance to cytotoxic substances or minimal media, cytotoxic orpro-apoptotic proteins, and proteins altering the growth or morphologyof the cell in which they are expressed; and wherein the part of the NIaprotease in a1) and the part of the NIa protease in a2) are generated bydividing the functional NIa protease at a position between amino acids60 and 80, wherein the NIa protease comprises an amino acid sequencehaving a catalytic triade comprising histidine at position 46, aspartateat position 81 and cysteine at position
 151. 2. A method of detectingand analyzing protein interactions in a cell, which comprises the steps:a) expressing in the cell a1) a first fusion protein comprising a firstinteraction partner and a part of the 27 kDa NIa protease of tobaccoetch virus, wherein the part of the NIa protease alone does not haveproteolytic activity, and a2) a second fusion protein comprising asecond interaction partner and another part of said NIa protease,wherein said another part of the NIa protease alone does not haveproteolytic activity, and a3) a reporter protein, whose reporteractivity can be activated or inactivated by proteolysis, b)reconstituting the functional NIa protease activity via interaction ofsaid first and second interaction partners, c) detecting and analyzingactivation of the proteolysis-activatable or inactivation of theproteolysis-inactivatable reporter protein by the reconstitutedfunctional NIa protease of step b), wherein said reporter protein isselected from the group consisting of recombinases, transcriptionactivators, fluorescent proteins, luciferases, beta-galactosidase,alkaline phosphatases, beta-lactamase, proteins and enzymes conferringresistance to cytotoxic substances or minimal media, cytotoxic orpro-apoptotic proteins, and proteins altering the growth or morphologyof the cell in which they are expressed; and wherein the part of the NIaprotease in a1) and the part of the NIa protease in a2) are the NIaprotease fragments Glyl-Thr70and Thr71-Gly243, respectively, wherein theNIa protease comprises an amino acid sequence having a catalytic triadecomprising histidine at position 46, aspartate at position 81 andcysteine at position
 151. 3. A method of detecting and analyzing proteininteractions in a cell, which comprises the steps: a) expressing in thecell a1) a first fusion protein comprising a first interaction partnerand a part of the 27 kDa NIa protease of tobacco etch virus, wherein thepart of the NIa protease alone does not have proteolytic activity, anda2) a second fusion protein comprising a second interaction partner andanother part of said NIa protease, wherein said another part of the NIaprotease alone does not have proteolytic activity, and a3) a reporterprotein, whose reporter activity can be activated or inactivated byproteolysis, b) reconstituting the functional NIa protease activity viainteraction of said first and second interaction partners, c) detectingand analyzing activation of the proteolysis-activatable or inactivationof the proteolysis-inactivatable reporter protein by the reconstitutedfunctional NIa protease of step b), wherein said reporter protein isselected from the group consisting of recombinases, transcriptionactivators, fluorescent proteins, luciferases, beta-galactosidase,alkaline phosphatases, beta-lactamase, proteins and enzymes conferringresistance to cytotoxic substances or minimal media, cytotoxic orpro-apoptotic proteins, and proteins altering the growth or morphologyof the cell in which they are expressed; and wherein the part of the NIaprotease in a1) and the part of the NIa protease in a2) are the NIaprotease fragements Glyl-Thr118 and Lys119-Gly243, respectively, whereinthe NIa protease comprises an amino acid sequence having a catalytictriade comprising histidine at position 46, aspartate at position 81 andcysteine at position
 151. 4. A method of detecting and analyzing proteininteractions in a cell, which comprises the steps: a) expressing in thecell a1) a first fusion protein comprising a first interaction partnerand a part of the 27 kDa NIa protease of tobacco etch virus, wherein thepart of the NIa protease alone does not have proteolytic activity, anda2) a second fusion protein comprising a second interaction partner andanother part of said NIa protease, wherein said another part of the NIaprotease alone does not have proteolytic activity, and a3) a reporterprotein, whose reporter activity can be activated or inactivated byproteolysis, b) reconstituting the functional NIa protease activity viainteraction of said first and second interaction partners, c) detectingand analyzing activation of the proteolysis-activatable or inactivationof the proteolysis-inactivatable reporter protein by the reconstitutedfunctional NIa protease of step b), wherein said reporter protein isselected from the group consisting of recombinases, transcriptionactivators, fluorescent proteins, luciferases, beta-galactosidase,alkaline phosphatases, beta-lactamase, proteins and enzymes conferringresistance to cytotoxic.